Hyperthermostable protease gene

ABSTRACT

There are provided hyperthermostable proteases having an amino acid sequences represented by SEQ ID Nos. 1, 3 and 5 of the Sequence Listing or functional equivalents thereof and hyperthermostable protease genes encoding those hyperthermostable protease. There is also disclosed a process for preparation of a hyperthermostable protease by culturing a transformant containing the gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 10/800,684, filed Mar. 16,2004, which is a divisional of application Ser. No. 09/841,553, filedApr. 24, 2001, which is a divisional of application Ser. No. 08/894,818,now issued as U.S. Pat. No. 6,261,822, which is a 371 national stageapplication of PCT/JP96/03253, filed Nov. 7, 1996, the entire contentsof the prior applications being incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a hyperthermostable protease useful asan industrial enzyme, a gene encoding the same and a method forpreparation of the enzyme by the genetic engineering.

BACKGROUND ART

The proteases are the enzymes which cleave peptide bonds in theproteins, and a number of the proteases have been found in animals,plants and microorganisms. They are used not only as reagents forresearch works and medical supplies, but also in industrial fields suchas additives for detergents, food processing and chemical synthesisutilizing the reverse reactions, and it can be said that they are veryimportant enzymes from an industrial viewpoint. For proteases to be usedin industrial fields, since very high physical and chemical stabilitiesare required, in particular, enzymes having high thermostabilities arepreferred to use. At present, proteases predominantly used in industrialfields are those produced by bacteria of the genus Bacillus because theyhave relatively high thermostability.

However, enzymes having further superior properties are desired andactivities have been attempted to obtain enzymes from microorganismswhich grow at high temperature, for example, thermophiles of the genusBacillus.

On the other hand, a group of microorganisms, named ashyperthermophiles, are well adapted themselves to high temperatureenvironments and therefor they are expected to be a source supplyingvarious thermostable enzymes. It has been known that one of thesehyperthermophiles, Pyrococcus furiosus, produces proteases [Appl.Environ. Microbiol., volume 56, page 1992-1998 (1990), FEMS Microbiol.Letters, volume 71, page 17-20 (1990) J. Gen. Microbiol., volume 137,page 1193-1199 (1991)].

A hyperthermophile belonging to the genus Pyrococcus, Pyrococcus sp.Strain KOD1 is reported to produce a thiol protease (cysteine protease)[Appl. Environ. Microbiol., volume 60, page 4559-4566 (1994)]. Bacteriabelonging to the genus Thermococcus, Staphylothermus andThermobacteroides, which are also hyperthermophiles, are known toproduce a protease [Appl. Microbiol. Biotechnol., volume 34, page715-719 (1991)].

OBJECTS OF THE INVENTION

As the proteases produced by these hyperthermophiles have highthermostabilities, they are expected to be applicable to newapplications to which any known enzymes has not been utilized. However,the above publication merely teach that thermostable protease activitiespresent in cell-free extract or crude enzyme solution obtained fromculture supernatant, and there is no disclosure about properties ofisolated and purified enzymes and the like. Only a protease produced bystrain KOD1 is obtained as the purified form. However, since a cysteineprotease has the defect that it easily loses the activity by oxidation,it is disadvantageous in the industrial use. In addition, since acultivation of microorganisms at high temperature is required to obtainenzymes from these hyperthermophiles, there is a problem in industrialproduction of the enzymes.

In order to solve the above problems, an object of the present inventionis to provide a protease of the hyperthermophiles which is advantageousin the industrial use, to isolate a gene encoding a protease of thehyperthermophiles, and to provide a method for preparation of ahyperthermostable protease using the gene by the genetic engineering.

DISCLOSURE OF THE INVENTION

In order to obtain a hyperthermostable protease gene, the presentinventors originally tried to purify a protease from microbial cells anda culture supernatant of Pyrococcus furiosus DSM3638 so as to determinea partial amino acid sequence of the enzyme. However, purification ofthe protease was very difficult in either cases of using the microbialcells or the culture supernatant, and the present inventors failed toobtain such an enzyme sample having sufficient purity for determinationof its partial amino acid sequence.

As a method for cloning a gene for an objective enzyme without anyinformation about a primary structure of the enzyme protein, there is anexpression cloning method. For example, a pullulanase gene originatingin Pyrococcus woesei (WO92/02614) has been obtained according to thismethod. However, in an expression cloning method, a plasmid vector isgenerally used and, in such case, it is necessary to use restrictionenzymes which can cleave an objective gene into relatively small DNAfragments so that the fragments can be inserted into the plasmid vectorwithout cleavage of any internal portion of the objective gene.Therefore, the expression cloning method is not always applicable tocloning of all kind of enzyme genes. Furthermore, it is necessary totest for an enzyme activity of a large number of clones and thisoperation is complicated.

The present inventors have attempted to isolate a protease gene by usinga cosmid vector which can maintain a larger DNA fragment (30-50 kb)instead of a plasmid vector to prepare a cosmid library of Pyrococcusfuriosus genome and investigating cosmid clone in the library to findout a clone expressing a protease activity. By using the cosmid vector,the number of transformants to be screened can be reduced in addition tolowering of possibilities of cleavage of a internal portion of theenzyme gene. On the other hand, since the copy number of a cosmid vectorin a host cell is not higher than that of a plasmid vector, it may bethat an amount of the enzyme expressed is too small to detect it.

In view of high thermostability of the objective enzyme, firstly, thepresent inventors have cultured respective transformants in a cosmidlibrary, separately, and have combined this step with a step forpreparing lysates containing only thermostable proteins from themicrobial cells thus obtained, and used these lysates for detecting theenzyme activity. Further, the use of the gelatin-containingSDS-polyacrylamide gel electrophoresis for detecting the proteaseactivity allowed the detection of a trace amount of the enzyme activity.

Thus, the present inventors obtained several cosmid clones expressingthe protease activity from the cosmid library of Pyrococcus furiosus andsuccessfully isolated a gene encoding a protease from the inserted DNAfragment contained in the clones. In addition, the present inventorsconfirmed that a protease encoded by the gene has the extremely highthermostability.

By comparing an amino acid sequence of the hyperthermostable proteasededuced from the nucleotide sequence of the gene with amino acidsequences of known proteases originating in microorganisms, homology ofthe amino acid sequence of the front half portion of thehyperthermostable protease with those of a group of alkaline serineproteases, a representative of which is subtilisin, has been shown. Inparticular, the extremely high homology has been found at each regionaround the four amino acid residues which are known to be important forthe catalytic activity of the enzyme. Thus, since the protease producedby Pyrococcus furiosus, which is active at such a high temperature thatproteases originating in mesophiles are readily inactivated, has beenshown to retain a structure similar to those of enzymes from mesophiles,it has been suggested that similar proteases would also be produced byhyperthermophiles other than Pyrococcus furiosus.

Then, the present inventors have noted possibilities that, in thenucleotide sequence of the hyperthermostable protease gene obtained, thenucleotide sequence encoding regions showing high homology withsubtilisin and the like can be used as a probe for detectinghyperthermostable protease gene, and have attempted to detect proteasegenes originating in hyperthermophiles by PCR using synthetic DNAsdesigned based on the nucleotide sequences as primers so as to clone thegene. As a result, it was found that a fragment of gene having thehomology with the above gene existed in a hyperthermophile, Thermococcusceler DSM2476. The cloning of the full length of the gene was difficultand this was thought to be due to that the product derived from the genewas harmful to the host.

The present inventors used Bacillus subtilis as a host for cloning andfound that harbouring of the full length gene was possible and theexpressed protease was extracellularly secreted, further revealed thatthe expressed protease showed the protease activity at 95° C. and hadthe high thermostability. Upon this, the molecular weight of a proteaseencoded by the gene was found to be less than half of that of thehigh-molecular protease derived from the Pyrococcus furiosus describedabove.

In addition, by hybridization using a fragment of the gene as a probe,we found that the second protease gene different from that of thehigh-molecular protease was present in Pyrococcus furiosus. The geneencodes a protease having a similar molecular weight to that of thehyperthermostable protease derived from Thermococcus celer, and the genewas isolated and introduced into Bacillus subtilis and, thereby, aproduct expressed from the gene was extracellularly secreted. Theexpressed protease showed the enzyme activity at 95° C. and had the highthermostability. In addition, the amino acid sequence of a matureprotease produced by processing of the protease was revealed.

As these two kinds of proteases are extracellularly secreted without anyspecial procedures, it is thought that a signal peptide encoded by thegene itself functions in Bacillus subtilis. The amount of expressed bothproteases per culture is remarkably higher as compared with thehigh-molecular protease derived from Pyrococcus furiosus which isexpressed in Escherichia coli or Bacillus subtilis. In addition, whenthe gene is expressed by utilizing a promoter of the subtilisin gene anda signal sequence, the amount of the expressed protease was furtherincreased.

Furthermore, the present inventors prepared a hybrid gene encoding ahybrid protease, i.e., a fusion protein from both proteases, andconfirmed that an enzyme expressed by hybrid gene showed the proteaseactivity at high temperature like the above hyperthermostable protease.

SUMMARY OF THE INVENTION

The first aspect of the present invention provides a hyperthermostableprotease having the amino acid sequence described in SEQ ID No. 1 of theSequence Listing or functional equivalents thereof as well as ahyperthermostable protease gene encoding the hyperthermostableproteases, inter alia, a hyperthermostable protease gene having thenucleotide sequence described in SEQ ID No. 2 of the Sequence Listing.Further, a gene which hybridizes with this hyperthermostable proteasegene and encodes a hyperthermostable protease is also provided.

In addition, the second aspect of the present invention provides ahyperthermostable protease having the amino acid sequence described inSEQ ID No. 3 of the Sequence Listing or functional equivalents thereofas well as a hyperthermostable protease gene encoding thehyperthermostable proteases, inter alia, a

hyperthermostable protease gene having the nucleotide sequence describedin SEQ ID No. 4 of the Sequence Listing. Further, a gene whichhybridizes with this hyperthermostable protease gene and encodes ahyperthermostable protease is also provided.

In addition, the third aspect of the present invention provides ahyperthermostable protease having the amino acid sequence described inSEQ ID No. 5 of the Sequence Listing or functional equivalents thereofas well as a hyperthermostable protease gene encoding thehyperthermostable proteases, inter alia, a

hyperthermostable protease gene having the nucleotide sequence describedin SEQ ID No. 6 of the Sequence Listing. Further, a gene whichhybridizes with this hyperthermostable protease gene and encodes ahyperthermostable protease is also provided.

Further, the present invention provides a method for preparation of thehyperthermostable protease which comprises cultivating a transformantcontaining the hyperthermostable protease gene of the present invention,and collecting the hyperthermostable protease from the culture.

As used herein, the term “functional equivalents” means as follows:

It is known that although, among naturally-occurring proteins, amutation such as deletion, addition, substitution and the like of one ora few (for example, up to 5% of the whole amino acids) amino acid(s) canoccur in the amino acid sequence thereof due to the modificationreaction and the like of the produced proteins in the living body orduring purification besides the polymorphism or mutation of the genesencoding them, there are proteins, in spite of the mutation describedabove, showing the substantially equivalent physiological or biologicalactivity to that of the proteins having no mutation. When the proteinshave the slight difference in the structures and, nevertheless, thegreat difference in the functions thereof is not recognized, they arecalled functional equivalents. This is true when the above mutations areartificially introduced into the amino acid sequence of the proteinsand, in this case, further a more variety of mutants can be made. Forexample, a polypeptide where a certain cysteine residue is replaced withserine residue in the amino acid sequence of human interleukin-2 (IL-2)shows the interleukin-2 activity [Science, volume 224, page 1431(1984)].

A product of the gene which is transcribed and translated from thehyperthermostable protease gene of the present invention is an enzymeprecursor (preproenzyme) containing two regions, one of them is a signalpeptide necessary for extracellular secretion and the other is apropeptide which is removed upon expression of the activity. When atransformant to which the above gene has been transferred can cleavethis signal peptide, an enzyme precursor (proenzyme) from which thesignal peptide has been removed is extracellularly secreted. Further, anactive form enzyme from which the propeptide has been removed isproduced by the self-digestion reaction between proenzymes. All of thepreproenzyme, proenzyme and active form enzyme thus obtained from thegene of the present invention are proteins which finally have theequivalent function and fall within the scope of “functionalequivalents”.

As apparent to those skilled in the art, an appropriate signal peptidemay be selected depending upon a host used for the expression of a geneof interest. The signal peptide may be removed when the extracellularsecretion is not desired. Therefore, among hyperthermostable proteasegenes disclosed herein, the genes from which a portion encoding a signalpeptide has been removed, and the genes where the portion is replacedwith other nucleotide sequence are also within the scope of the presentinvention in the context that they encode the proteases showing theessentially equivalent activity.

As used herein, a gene which “hybridizes to a hyperthermostable proteasegene” refers to a gene which hybridizes with the hyperthermostableprotease gene under the stringent conditions, that is, those whereincubation is carried out at 50° C. for 12 to 20 hours in 6×SSC (1×SSCrepresents 0.15M NaCl, 0.015M sodium citrate, pH7.0) containing 0.5%SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1%Ficoll 400 and 0.01% denatured salmon sperm DNA.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a figure showing a restriction map of a DNA fragment derivedfrom Pyrococcus furiosus contained in the plasmid pTPR12 and the plasmidpUBP13.

FIG. 2 is a figure showing a design of the oligonucleotide PRO-1F (SEQID NO:9) based on nucleotides 628 to 669 of SEQ ID NO:7 which encoderesidues 169 to 182 of SEQ ID NO:8.

FIG. 3 is a figure showing a design of the oligonucleotide PRO-2F (SEQID NO:10) based on nucleotides 1210 to 1251 of SEQ ID NO:7 which encoderesidues 363 to 376 of SEQ ID NO:8 and PRO-2R (SEQ ID NO:11).

FIG. 4 is a figure showing a design of the oligonucleotide PRO-4R (SEQID NO:12) based on nucleotides 1882 to 1923 of SEQ ID NO:7 which encoderesidues 587 to 600 of SEQ ID NO:8.

FIG. 5 is a restriction map of the plasmid p2F-4R.

FIG. 6 is a restriction map of the plasmid pTC3.

FIG. 7 is a restriction map of the plasmid pTCS6.

FIG. 8 is a restriction map of the plasmid pTC4.

FIG. 9 is a figure showing the procedures for constructing the plasmidpSTC3.

FIG. 10 is a restriction map of the plasmid pSTC3.

FIG. 11 is a figure comparing the amino acid sequences of the variousproteases of PFUL (SEQ ID NO:8), TCES (SEQ ID NO:1) and Subtilisin (SEQID NO:45).

FIG. 12 is a continuation of FIG. 11.

FIG. 13 is a figure showing a restriction map around the protease PFUSgene on the Pyrococcus furiosus chromosomal DNA.

FIG. 14 is a restriction map of the plasmid pSPT1.

FIG. 15 is a restriction map of the plasmid pSNP1.

FIG. 16 is a restriction map of the plasmid pPS1.

FIG. 17 is a restriction map of the plasmid pNAPS1.

FIG. 18 is a figure showing the optimum temperature for the enzymepreparation TC-3.

FIG. 19 is a figure showing the optimum temperature for the enzymepreparation NAPS-1.

FIG. 20 is a figure showing the optimum pH for the enzyme preparationTC-3.

FIG. 21 is a figure showing the optimum pH for the enzyme preparationNP-1.

FIG. 22 is a figure showing the optimum pH for the enzyme preparationNAPS-1.

FIG. 23 is a figure showing the thermostability of the enzymepreparation TC-3.

FIG. 24 is a figure showing the thermostability of the enzymepreparation NP-1.

FIG. 25 is a figure showing the thermostability of the activated enzymepreparation NP-1.

FIG. 26 is a figure showing the thermostability of the enzymepreparation NAPS-1.

FIG. 27 is a figure showing the pH-stability of the enzyme preparationNP-1.

FIG. 28 is a figure showing the stability of the enzyme preparation NP-1in the presence of SDS.

FIG. 29 is a figure showing the stability of the enzyme preparationNAPS-1 in the presence of SDS.

FIG. 30 is a figure showing the stability of the enzyme preparationNAPS-1 in the presence of acetonitrile.

FIG. 31 is a figure showing the stability of the enzyme preparationNAPS-1 in the presence of urea.

FIG. 32 is a figure showing the stability of the enzyme preparationNAPS-1 in the presence of guanidine hydrochloride.

PREFERRED EMBODIMENTS OF THE INVENTION

The hyperthermostable protease gene of the present invention can beobtained by screening the gene library of hyperthermophiles. As thehyperthermophile, bacteria belonging to the genus Pyrococcus can be usedand the gene of interest can be obtained by screening a cosmid libraryof Pyrococcus furiosus genome.

For example, Pyrococcus furiosus DSM3638 can be used as Pyrococcusfuriosus, and the strain is available from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH.

One example of the cosmid libraries of Pyrococcus furiosus genome can beobtained by ligating DNA fragments which are obtained by partialdigestion of the genomic DNA of Pyrococcus furiosus DSM3638 with arestriction enzyme Sau3A1 (manufactured by Takara Shuzo Co., Ltd.), withthe triple helix cosmid vector (manufactured by Stratagene), andpackaging the ligated product into a lambda phage particle according tothe in vitro packaging method. Then, the library is transduced into thesuitable Escherichia coli, for example, Escherichia coli DH5αMCR(manufactured by BRL) to obtain the transformants, followed by

cultivation them, collecting the microbial cells, subjecting them toheat treatment (for example, 100° C. for 10 minutes), sonicating andsubjecting them to heat treatment (for example, 100° C. for 10 minutes)again. The presence or absence of the protease activity in the resultinglysate can be screened by utilizing the gelatin-containingSDS-polyacrylamide gel electrophoresis.

In this manner, a cosmid clone containing a hyperthermostable proteasegene expressing a protease which is resistant to the above heattreatment can be obtained.

Further, the cosmid DNA prepared from the cosmid clone thus obtained canbe digested into fragments with a suitable restriction enzyme to obtaina recombinant plasmid with each fragment incorporated. Then, a suitablemicroorganism is transformed with the plasmid, and the protease activityexpressed by the resulting transformant can be examined to obtain arecombinant plasmid containing a hyperthermostable protease gene ofinterest.

That is, the cosmid prepared from one of the above cosmid clones isdigested with NotI and PvuII (both manufactured by Takara Shuzo Co.,Ltd.) to give an about 7.5 kb DNA fragment which can be isolated andinserted between the NotI site and the SmaI site of the plasmid vectorpUC19 (manufactured by Takara Shuzo Co., Ltd.) into which the NotIlinker (manufactured by Takara Shuzo Co., Ltd.) has been introduced. Theplasmid was designated the plasmid pTPR12 and Escherichia coli JM109transformed with the plasmid was designated Escherichia coliJM109/pTPR12 and has been deposited at National Institute of Bioscienceand Human-Technology at 1-1-3. Higashi, Tsukuba-shi, Ibaraki-ken, Japansince May 24, 1994 (original deposit date) as the accession number FERMBP-5103 under Budapest Treaty.

The lysate of the Escherichia coli JM109/pTPR12 shows the similarprotease activity to that of the above cosmid clone on thegelatin-containing SDS-polyacrylamide gel.

The nucleotide sequence of the DNA fragment, derived from Pyrococcusfuriosus, which was inserted into the plasmid pTPR12 can be determinedby a conventional method, for example, the dideoxy method. Thenucleotide sequence of the 4.8 kb portion flanked by two DraI siteswithin the DNA fragment inserted into the plasmid pTPR12 is shown in SEQID No. 7 of the Sequence Listing. The amino acid sequence of a geneproduct deduced from the nucleotide sequence is shown in SEQ ID No. 8 ofthe Sequence Listing. Thus, a hyperthermostable protease, the nucleotidesequence and the amino acid sequence of which were revealed, derivedfrom Pyrococcus furiosus was designated the protease PFUL. As shown inSEQ ID No. 8 of the Sequence Listing, the protease PFUL is a proteaseconsisting of 1398 residues and having a high-molecular weight of morethan 150 thousands.

The protease PFUL gene can be expressed using Bacillus subtilis as ahost. As Bacillus subtilis, Bacillus subtilis DB104 can be used and thestrain is the known one described in Gene, volume 83, page 215-233(1989). As a cloning vector, the plasmid pUB18-P43 can be used and theplasmid was gifted from Dr. Sui-Lam Wong at Calgary University. Theplasmid contains the kanamycin resistant gene as a selectable marker.

There is the plasmid pUBP13 where an about 4.8 kb DNA fragment obtainedby digestion of the plasmid pTPR13 with DraI has been inserted into theSmaI site of the plasmid vector pUB18-P43. In the plasmid, the proteasePFUL gene is located downstream of the P43 promoter [J. Biol. Chem.,volume 259, page 8619-8625 (1984)] which functions in Bacillus subtilis.Bacillus subtilis DB104 transformed with the plasmid was designatedBacillus subtilis DB104/pUBP13. The lysate of the transformant shows thesimilar protease activity to that of the

Escherichia coli JM109/pTPR12.

However, only a trace amount of the protease activity is detected in aculture supernatant of the transformant. This is thought to be due tothat a molecular weight of the protease PFUL is extremely high and it isnot translated effectively in Bacillus subtilis, and that a signalsequence encoded by the protease PFUL gene dose not function well inBacillus subtilis. There is a possibility that the protease PFUL is amembrane-bound type protease, and the peptide chain on the C-terminalside of the protease PFUL may be a region for binding to the cellmembrane.

FIG. 1 shows a restriction map around the protease PFUL gene on thePyrococcus furiosus chromosome, as well as a DNA fragment inserted intothe plasmid pTPR12 and that inserted into the plasmid pUBP13. Inaddition, an arrow in FIG. 1 shows the open reading frame encoding theprotease PFUL.

By comparing the amino acid sequence of the protease PFUL represented bySEQ ID. No. 8 of the Sequence Listing with that of a protease derivedfrom the known microorganism, it is seen that there is the homologybetween the amino acid sequence of the front half portion of theprotease PFUL and that of a group of alkaline serine proteases, arepresentative of which is subtilisin [Protein Engineering, volume 4,page 719-737 (1991)], and that there is the extremely high homologyaround four amino acid residues which are considered to be important forcatalytic activity of the proteases.

As it was revealed that regions commonly present in the proteasesderived from a mesophile are conserved in the amino acid sequence of theprotease PFUL produced by the hyperthermophile Pyrococcus furiosus, itis expected that these regions are present in the same kind of proteasesproduced by the hyperthermophiles other than Pyrococcus furiosus.

That is, a DNA having the suitable length can be prepared based on thesequence of a portion encoding the amino acid sequence of a regionhaving the high homology with that of subtilisin and the like, and theDNA can be used as a probe for hybridization or as a primer for geneamplification such as PCR and the like to screen a hyperthermostableprotease gene similar to the present enzyme present in varioushyperthermophiles.

In the above method, a DNA fragment containing only a portion of thegene of interest is obtained in some cases. Upon this, the nucleotidesequence of the resulting DNA fragment is investigated and confirmedthat it is a portion of the gene of interest and, thereafter,hybridization can be carried out using the DNA fragment or a partthereof as a probe or PCR can be carried out using a primer synthesizedbased on the nucleotide sequence of the DNA fragment to obtain the wholegene of interest.

The above hybridization can be carried out under the followingconditions. That is, a membrane to which a DNA is fixed is incubatedwith a probe suitably labeled at 50° C. for 12 to 20 hours in 6×SSC(1×SSC represents 0.15M NaCl, 0.015M sodium citrate, pH 7.0) containing0.5% SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone,0.1% Ficoll 400 and 0.01% denatured salmon sperm DNA. After thecompletion of incubation, the membrane is washed, beginning with washingat 37° C. in 2×SSC containing 0.5% SDS, varying the SSC concentration ina range of to 0.1× and a temperature in a range of to 50° C., until asignal from a probe hybridized to the fixed DNA can be discriminatedfrom the background.

In addition, it is apparent to those skilled in the art that a probe anda primer can be made based on the thus obtained new hyperthermostablegene to obtain another hyperthermostable protease gene according to thesimilar method.

FIGS. 2, 3 and 4 show the relationship among the amino acid sequences ofregions in the amino acid sequence of the protease PFUL which have highhomology with those of subtilisin and the like, the nucleotide sequenceof the protease PFUL gene encoding the region, and the nucleotidesequences of the oligonucleotides PRO-1F, PRO-2F, PRO-2R and PRO-4Rwhich were synthesized based thereon. Further, SEQ ID Nos. 9, 10, 11 and12 of the Sequence Listing show the nucleotide sequences of theoligonucleotides PRO-1F, PRO-2F, PRO-2F and PRO-4R. That is, SEQ ID Nos.9-12 are the nucleotide sequences of one example of the oligonucleotidesused for screening the hyperthermostable protease gene of the presentinvention.

By using a combination of the oligonucleotides as primer, a proteasegene can be detected by PCR using a chromosomal DNA of the varioushyperthermophiles as a template.

As the hyperthermophiles, the bacteria belonging to the genusPyrococcus, genus Thermococcus, genus Staphylothermus, genusThermobacteroides and the like can be used. As the bacteria belonging togenus Thermococcus, for example, Thermococcus celer DSM2476 can be usedand the strain can be obtained from Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH. When PCR is carried out using achromosomal DNA of Thermococcus celer DSM2476 as a template and using acombination of the oligonucleotides PRO-1F and PRO-2R or a combinationof the oligonucleotides PRO-2F and PRO-4R as a primer, the specificamplification of a DNA fragment is observed and the presence of aprotease gene can be identified. In addition, the amino acid sequenceencoded by the DNA fragment can be estimated by inserting the DNAfragments into a suitable plasmid vector to make a recombinant plasmidand, thereafter, determining the nucleotide sequence of the inserted DNAfragment by the dideoxy method.

A DNA fragments of about 150 bp amplified using the oligonucleotidesPRO-1F and PRO-2R and DNA fragment of about 550 bp DNA amplified usingthe oligonucleotides PRO-2F and PRO-4R are inserted into the HincII siteof the plasmid vector pUC18 (manufactured by Takara Shuzo Co., Ltd.).The recombinant plasmids are designated the plasmid p1F-2R(2) and theplasmid p2F-4R, respectively. SEQ ID No. 13 of the Sequence Listingshows the nucleotide sequence of the inserted DNA fragment in theplasmid p1F-2R(2) and the amino acid sequence deduced therefrom and SEQID No. 14 of the Sequence Listing shows the nucleotide sequence of theinserted DNA fragment in the plasmid p2F-4R and the amino acid sequencededuced therefrom. In the SEQ ID No. 13 of the Sequence Listing, thenucleotide sequence from the 1st to the 21st nucleotides and that fromthe 113rd to the 145th nucleotides and, In the SEQ ID No. 14 of theSequence Listing, the nucleotide sequence from the 1st to the 32ndnucleotides and that from the 532nd to the 564th nucleotides are thenucleotide sequence derived from the oligonucleotides used in PCR asprimers (each corresponding to the oligonucleotides PRO-1F, PRO-2R,PRO-2F and PRO-4R, respectively). The amino acid sequences having thehomology with that of the protease PFUL and the alkaline serineproteases derived from the various microorganisms are present in theamino acid sequences represented by SEQ ID Nos. 13 and 14 of theSequence Listing, indicating that the above PCR-amplified DNA fragmentswere amplified with the protease gene as a template.

A restriction map of the plasmid p2F-4R is shown in FIG. 5. In FIG. 5, athick solid line indicates the DNA fragment inserted into the plasmidvector pUC18.

Then, a hyperthermostable protease gene, for example, a gene of thehyperthermostable protease produced by Thermococcus celer can beobtained by screening the gene library of hyperthermostable bacteriausing above oligonucleotides or the amplified DNA fragments obtained bythe above PCR as a probe.

One example of the gene libraries of Thermococcus celer, there is alibrary prepared by partially digesting a chromosomal DNA ofThermococcus celer DSM2476 with the restriction enzyme Sau3AI to obtaina DNA fragment, ligating the fragment with lambda GEM-11 vector(manufactured by Promega) and packaged it into the lambda phage particleusing the in vitro packaging method. Then, the library can be transducedinto suitable Escherichia coli, for example, Escherichia coli LE392(manufactured by Promega) to allow to form the plaques on a plate, andplaque hybridization can be carried out using an amplified DNA fragmentobtained by the above PCR as a probe to obtain phage clones containing agene of interest.

Further, a phage DNA prepared from the phage clones thus obtained can bedigested with a suitable restriction enzyme, and southern hybridizationcan be carried out using the above probe to detect a DNA fragmentcontaining a protease gene.

When the phage DNA prepared from the phage clones obtained by the plaquehybridization is digested with KpnI and BamHI (both manufactured byTakara Shuzo Co., Ltd.), an about 5 kb DNA fragment is hybridized to theprobe, and the about 5 kb DNA fragment can be isolated and insertedbetween the KpnI site and the BamHI site of the plasmid vector pUC119(manufactured by Takara Shuzo Co., Ltd.) to obtain a recombinantplasmid. The plasmid was designated the plasmid pTC3 and Escherichiacoli JM109 transformed with the plasmid was designated Escherichia coliJM109/pTC3. A restriction map of the plasmid pTC3 is shown in FIG. 6. InFIG. 6, a thick solid line designates the DNA fragment inserted into theplasmid vector pUC119.

A DNA fragment which does not contain the protease gene within the DNAfragment inserted into the plasmid pTC3 can be removed according to thefollowing procedures. That is, after the plasmid pTC3 is digested withSacI (manufactured by Takara Shuzo Co., Ltd.), southern hybridization iscarried out according to the similar procedures described above and itis found that an about 1.9 kb DNA fragment hybridizes to the probe.Then, the about 1.9 kb DNA fragment can be isolated and inserted intothe SacI site of the plasmid vector pUC118 (manufactured by Takara ShuzoCo., Ltd.) to make a recombinant vector. The plasmid was designated theplasmid pTCS6 and Escherichia coli JM109 transformed with the plasmidwas designated Escherichia coli JM109/pTCS6. A restriction map of theplasmid pTCS6 is shown in FIG. 7. In FIG. 7, a thick solid linedesignates the DNA fragment inserted into the plasmid vector pUC118. Bydetermining the nucleotide sequence of the DNA fragment inserted intothe plasmid pTCS6 by the dideoxy method, it can be confirmed that aprotease gene is present in the DNA fragment. SEQ ID No. 15 of theSequence Listing shows the nucleotide sequence of the fragment. Bycomparing the nucleotide sequence with that of the DNA fragment insertedinto the plasmid p1F-2R (2) or that of the plasmid p2F-4R represented bySEQ ID No. 13 or 14 of the Sequence Listing, it is seen that the DNAfragment inserted into the plasmid pTCS6 contains the DNA fragment whichis also shared by the plasmid p2F-4R but lacks a 5′ region of theprotease gene.

Like this, the hyperthermostable protease gene, derived fromThermococcus celer, contained in the plasmid pTCS6 lacks a portionthereof. However, as apparent to those skilled in the art, a DNAfragment covering the full length hyperthermostable protease gene can beobtained by (1) screening the gene library once more, (2) conductingsouthern hybridization using a chromosomal DNA, or (3) obtaining a DNAfragment of a 5′ upstream region by PCR using a cassette and a cassetteprimer (Takara Shuzo Co., Ltd., Genetic Engineering Products Guidance,1994-1995 edition, page 250-251).

The present inventors selected the method (3). That is, a chromosomalDNA of the Thermococcus celer is completely digested with a fewrestriction enzymes, followed by ligation with a cassette (manufacturedby Takara Shuzo Co., Ltd.) which corresponds to the

restriction enzyme used. PCR is carried out using this ligation productas a template and the primer TCE6R (SEQ ID No. 16 of the SequenceListing shows the nucleotide sequence of the primer TCE6R) and thecassette primer C1 (manufactured by Takara Shuzo Co., Ltd.) as primers.When the above procedures are carried out using the restriction enzymeHindIII (manufactured by Takara Shuzo Co., Ltd.), an about 1.8 kb DNAfragment is amplified, and a DNA fragment of about 1.5 kb which isobtained by digesting above amplified fragment with HindIII and SacI canbe inserted into between the HindIII site and the SacI site of theplasmid vector pUC119 to obtain a recombinant plasmid. The plasmid wasdesignated the plasmid pTC4 and Escherichia coli JM109 transformed withthe plasmid was designated Escherichia coli JM109/pTC4. A restrictionmap of the plasmid pTC4 is shown in FIG. 8. In FIG. 8, a thick solidline designates the DNA fragment inserted into the plasmid vectorpUC119.

By determining the nucleotide sequence of the DNA fragment inserted intothe plasmid pTC4 by the dideoxy method, it can be confirmed that aprotease gene is present in the DNA fragment. SEQ ID No. 17 of theSequence Listing shows the nucleotide sequence of the fragment. Bycomparing the amino acid sequence deduced from the nucleotide sequencewith those of the various proteases, it is found that the DNA fragmentinserted into the plasmid pTC4 covers the 5′ region of thehyperthermostable protease gene which the plasmid pTCS6 lacks. Bycombining the nucleotide sequence with that of the DNA fragment insertedinto the plasmid pTCS6 represented by SEQ ID No. 15 of the SequenceListing, the nucleotide sequence of the full length hyperthermostablegene derived from Thermococcus celer can be identified. The nucleotidesequence of the open reading frame present in the obtained nucleotidesequence is shown in SEQ ID No. 2 of the Sequence Listing and the aminoacid sequence deduced from the nucleotide sequence is shown in SEQ IDNo. 1, respectively. Thus, the hyperthermostable protease derived fromThermococcus celer, with the nucleotide sequence encoding it and theamino acid sequence thereof revealed was designated the protease TCES.The full length of the protease TCES gene can be reconstituted bycombining the inserted DNA fragment of the plasmid pTC4 and that of theplasmid pTCS6.

It is contemplated that the protease activity expressed by the gene canbe confirmed by reconstituting the full length protease TCES gene fromtwo DNA fragments contained in pTC4 and pTCS6, and inserting thisdownstream of the lac promoter of a plasmid to give an expressionplasmid which is introduced into Escherichia coli. However, this methodaffords no transformants into which the expression vector of interesthas been introduced, and it is predicted that the production of aproduct expressed from the gene is harmful or lethal to Escherichiacoli. It is contemplated that, in such as case, for example, a proteaseis extracellularly secreted using Bacillus subtilis as a host to confirmthe activity.

As a host for expressing the protease TCES gene in Bacillus subtilis,the Bacillus subtilis DB104 can be used and, as a cloning vector, theplasmid pUB18-P43 can be used.

However, since the host-vector system for Escherichia coli has theadvantages that it contains various kind of vectors and transformationcan be carried out simply and highly effectively, as many as possibleprocedures for constructing an expression vector are desirably, ifpossible, carried out by using Escherichia coli. That is, in Escherichiacoli, an optional nucleotide sequence containing a termination codon isinserted between two protease gene fragments derived from the plasmidpTC4 and the plasmid pTCS6 so that the full length protease TCES gene isnot reconstituted, thus, making expression of the gene productimpossible and, therefore, the construction of a plasmid can be carriedout. Then, this inserted sequence can be removed at the final stage tomake the expression plasmid pSTC3 of interest shown in FIG. 10.

The procedures for constructing the plasmid pSTC3 shown in FIG. 9 areexplained below.

First, the about 1.8 kb HindIII-SspI fragment inserted into the plasmidpTCS6 is inserted between the HindIII site and the EcoRV site of theplasmid vector pBR322 (manufactured by Takara Shuzo Co., Ltd.) to makethe recombinant plasmid pBTC5 and, from this plasmid, the DNA fragmentbetween the HindIII site and the KpnI site derived from a multicloningsite of the plasmid vector pUC118 and the BamHI site present on theplasmid vector pBR322 are removed to make the plasmid pBTC5HKB.

Then, based on the nucleotide sequence of the protease TCES gene, theprimer TCE12 which can introduce the EcoRI site and the BamHI site infront of an initiation codon of the protease TCES, and the primer TCE20Rwhich can introduce the ClaI site and a termination codon on the 3′ sideof only one SacI site present in the nucleotide sequence are synthesizedSEQ ID Nos. 18 and 19 of the Sequence Listing show the nucleotidesequences of the primer TCE12 and the primer TCE20R, respectively.

An about 0.9 kb DNA fragment which has been amplified by PCR using achromosomal DNA of Thermococcus celer as a template and using these twoprimers is digested with EcoRI and ClaI (manufactured by Takara ShuzoCo., Ltd.), and inserted into between the EcoRI site and the ClaI siteof the plasmid pBTC5HKB to obtain the plasmid pBTC6, which has a mutantgene where the nucleotide sequence of 69 bp long including a terminationcodon is inserted into the SacI site of the protease TCES gene.

A ribosome binding site derived from the Bacillus subtilis P43 promoter[J. Biol. Chem., volume 259, page 8619-8625 (1984)] is introducedbetween the KpnI site and the BamHI site of the plasmid vector pUC18 tomake the plasmid pUC-P43. The nucleotide sequences of the syntheticoligonucleotides BS1 and BS2 are shown in SEQ ID Nos. 20 and 21 of theSequence Listing, respectively. Then, the plasmid pBTC6 is digested withBamHI and SphI (both manufactured by Takara Shuzo Co., Ltd.) to obtainan about 3 kb DNA fragment containing a mutant gene of the proteaseTCES, which is inserted between the BamHI site and the SphI site of theplasmid pUC-P43 to construct the plasmid pTC12.

All the above procedures for constructing a plasmid can be carried outusing Escherichia coli as a host.

The SacI site present in the plasmid vector pUC18-P43 used for cloninginto Bacillus subtilis is previously removed, and an about 3 kbKpnI-SphI DNA fragment obtained from the pTC12 can be inserted intobetween the KpnI site and the SphI site to make the plasmid pSTC2 usingBacillus subtilis DB104 as a host. The plasmid contains a mutant gene ofthe protease TCES having the P43 promoter and a ribosome binding sitesequence on its 5′ side. After the plasmid pSTC2 is digested with SacI,and intramolecular ligation is carried out to obtain a

recombinant plasmid, from which the inserted sequence contained in theSacI site of the above mutant gene has been removed. The recombinantplasmid was designated the plasmid pSTC3, and Bacillus subtilis DB104transformed with the plasmid was designated Bacillus subtilisDB104/pSTC3 and has been deposited at National Institute of Bioscienceand Human-Technology at 1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japanunder accession number FERM BP-5635 since Dec. 1, 1995 (original depositdate) according to Budapest Treaty. The transformant is cultured, and aculture supernatant and an extract from the cells were investigated forthe protease activity. As a result, the thermostable protease activityis found in both samples.

FIG. 10 shows a restriction map of the plasmid pSTC3. In FIG. 10, athick solid line designates the DNA fragment inserted into the plasmidvector pUB18-P43.

When the amino acid sequences of the protease PFUL, the protease TCESand subtilisin are aligned so that the regions having the homologycoincide with each other as shown in FIGS. 11 and 12, it is seen thatthe protease PFUL has the regions which is not homologous with thesequence of the protease TCES and that of subtilisin at the C-terminalside thereof as well as between the regions having the homology. Fromthis, it is contemplated that, besides the protease PFUL, a proteasehaving a smaller molecular weight than that of the protease PFUL, suchas the protease TCES or subtilisin may be present in

Pyrococcus furiosus. In order to search a gene encoding such a protease,southern hybridization can be carried out using a chromosomal DNAprepared from Pyrococcus furiosus as a target, and using a DNA fragmentcontaining the nucleotide sequence within the protease TCES gene, whichencoding the amino acid sequence which is well conserved in threeproteases, for example, the about 150 bp DNA fragment inserted into theplasmid p1F-2R (2), as a probe.Although, since the DNA fragment used for a probe has also the homologywith the protease PFUL gene, the gene fragment is detected as a signaldepending upon the hybridization conditions, the position of the signalderived from the gene can be previously estimated on each restrictionenzyme used for cutting a chromosomal DNA, from the informations on thenucleotide sequence of the protease PFUL gene and the restriction map.When some enzymes are used, in addition to the position predicted on theprotease PFUL gene, an another signal is detected as almost the samelevel, suggesting the possibility that at least one protease is presentin Pyrococcus furiosus in addition to the protease PFUL.

For isolating a gene corresponding to the above new signal, a portion ofthe gene is first cloned so as to prevent the failure of isolation ofthe gene, as in a case of the protease TCES, resulted from theexpression of the gene product which is harmful or lethal to Escherichiacoli. For example, when a chromosomal DNA of Pyrococcus furiosus isdigested with the restriction enzymes SacI and SpeI (both manufacturedby Takara Shuzo Co., Ltd.) and the digestion products are used toconduct southern hybridization as described above using a fragment ofthe protease TCES gene as a probe, it was revealed that a new signalcorresponding to about 0.6 kb, derived from the new gene, was observedreplacing with a signal corresponding to about 3 kb which was observedin a case of digestion with only SacI. This about 0.6 kb SpeI-SacIfragment encodes the amino acid sequence of at maximum around 200residues and it cannot be contemplated to express a protease having theactivity. A Pyrococcus furiosus chromosomal DNA digested with SacI andSpeI is subjected to agarose gel electrophoresis to recover a DNAfragment corresponding to about 0.6 kb from the gel.

Then, the fragment is inserted between the SpeI site and the SacI siteof the plasmid vector pBluescript SK(−) (manufactured by Stratagene) andthe resulting recombinant plasmid is used to transform Escherichia coliJM109. From this transformant, a clone with a fragment of interestincorporated can be obtained by colony hybridization using the sameprobe as that used for the above southern hybridization. Whether aplasmid contained in the resulting clone has the sequence encoding aprotease or not can be confirmed by conducting PCR using the primers1FP1, 1FP2, 2RP1 and 2RP2 (the nucleotide sequences of the primers 1FP1,1FP2, 2RP1 and 2RP2 are shown in SEQ ID Nos. 22, 23, 24, and 25 of theSequence Listing) made based on the amino acid sequence common to theabove various proteases, or by determining the nucleotide sequence of aDNA fragment inserted into the plasmid prepared from the clone. Theplasmid in which the existence of a protease gene is confirmed in thismanner was designated the plasmid pSS3. The nucleotide sequence of a DNAfragment inserted in the plasmid, and the amino acid sequence deducedtherefrom are shown in SEQ ID No. 26 of the Sequence Listing.

The amino acid sequence deduced from the nucleotide sequence of the DNAfragment inserted into the plasmid pSS3 is shown to have the homologywith the sequences of subtilisin, the protease PFUL, the protease TCESand the like. A product of a protease gene different from the proteasePFUL, a portion of which was obtained newly from Pyrococcus furiosus,was designated the protease PFUS. A region encoding a N-terminal sidepart of the protease, that is, a region 5′ of the SpeI site, and aregion encoding a C-terminal side part, that is, a gene fragment 3′ ofthe above SacI site can be obtained by the inverse PCR method. If therestriction enzyme sites in the protease PFUS gene and the vicinitythereof in a chromosome are revealed in advance, the inverse PCR can becarried out using an appropriate restriction enzyme. The restrictionenzyme sites can be revealed by cutting a chromosomal DNA of Pyrococcusfuriosus with various restriction enzymes, and conducting southernhybridization using a DNA fragment inserted into the plasmid pSS3 as aprobe. Consequently, it is shown that the SacI site is located on about3 kb distant 5′ side of the SpeI site and the XbaI site is located onabout 5 kb distant 3′ side of the SacI site.

A primer used for the inverse PCR can be design to anneal at around anend of the SpeI-SacI fragment contained in the plasmid pSS3. The primersdesigned to anneal at around the SacI site are designated NPF-1 andNPF-2 and a primer designed to anneal at around the SpeI site isdesignated NPR-3. The nucleotide sequences thereof are shown in SEQ IDNos. 27, 28 and 29 of the Sequence Listing, respectively.

A chromosomal DNA of Pyrococcus furiosus is digested with SacI or XbaI(both manufactured by Takara Shuzo Co., Ltd.), respectively, which isallowed to intramolecullarly ligate, and this reaction mixture can beused as a template for the inverse PCR. When a chromosomal DNA isdigested with SacI, an about 3 kb fragment is amplified by the inversePCR, which is inserted into the plasmid vector pT7BlueT (manufactured byNovagen) to obtain a recombinant plasmid which was designated theplasmid pS322. On the other hand, in a case of a chromosomal DNAdigested with XbaI, an about 9 kb fragment is amplified. The amplifiedfragment is digested with XbaI to obtain an about 5 kb fragment which isrecovered and inserted into the plasmid vector pBluescript SK(−) toobtain a

recombinant plasmid, which was designated the plasmid pSKX5. Bycombining the results of southern hybridization performed using theSpeI-SacI fragment contained in the plasmid pSS3 as a probe, and thoseof analysis on the plasmids pS322 and pSKX5 with the restrictionenzymes, a restriction map of the protease PFUS gene and the vicinitythereof in a chromosome can be obtained. The restriction map is shown inFIG. 13.

In addition, by analyzing the nucleotide sequence on a 5′ fragmentinserted into the plasmid pS322 in a 5′ direction starting from the SpeIsite, the amino acid sequence of an enzyme protein encoded by the regioncan be deduced. The resulting nucleotide sequence and the amino acidsequence deduced therefrom are shown in SEQ ID No. 30 of the SequenceListing. Since the amino acid sequence of this region has the homologywith that of a protease such as subtilisin or the like, an initiationcodon of the protease PFUS can be presumed based on this homology and,thus, primer NPF-4 which can introduce the BamHI site in front of theinitiation codon of the protease PFUS can be designed. On the otherhand, the nucleotide sequence determined by analyzing the nucleotidesequence of a 3′ fragment of the protease PFUS gene inserted into theplasmid pSKX5 in a 5′ direction starting from the XbaI site is shown inSEQ ID No. 31 of the Sequence Listing. Based on the nucleotide sequence,the primer NPR-4 which can insert the SphI site into the vicinity of theXbaI site can be designed. The nucleotide sequences of the primers NPF-4and NPR-4 are shown in SEQ ID Nos. 32 and 33 of the Sequence Listing,respectively. The full length protease PFUS gene can be amplified byusing these two primers and using a chromosomal DNA of Pyrococcusfuriosus as a template.

The protease PFUS can be expressed in the Bacillus subtilis system, asin a case of the protease TCES. A plasmid for expressing the proteasePFUS can be constructed based on the expression plasmid pSTC3 for theprotease TCES. First, a DNA fragment containing the full length proteasePFUS gene which can be amplified by the PCR is digested with BamHI andSacI to recover an about 0.8 kb fragment encoding a N-terminal part ofthe enzyme. And this fragment is replaced with the BamHI-SacI fragment,also encoding a N-terminal part of the protease TCES, of the plasmidpSTC3. The resulting expression plasmid encoding a hybrid protein of theprotease TCES and the protease PFUS gene was designated the plasmidpSPT1. FIG. 14 shows a restriction map of the plasmid pSPT1.

Then, the above PCR-amplified DNA fragment is digested with SpeI andSphI to give an about 5.7 kb fragment which is isolated and replacedwith the SpeI-SphI fragment encoding a C-terminal part of the proteaseTCES in the plasmid pSPT1. The expression plasmid thus constructed wasdesignated the plasmid pSNP1, and Bacillus subtilis DB104 transformedwith the plasmid was designated Bacillus subtilis DB104/pSNP1 and hasbeen deposited at National Institute of Bioscience and Human-Technology(NIBH) at 1-1-3, Higashi, Tsukuba-shi, Ibaraki-ken, Japan since Dec. 1,1995 (original deposit date) as the accession number FERM BP-5634 underBudapest Treaty. FIG. 15 shows a restriction map of the plasmid pSNP1.

The Bacillus subtilis DB104/pSNP1 is cultured and a culture supernatantand an extract from the cells are examined for the protease activity andit is found that the thermostable protease activity is found in bothsamples.

The nucleotide sequence of a gene encoding the protease PFUS can bedetermined by digesting a DNA fragment inserted into the plasmid pSNP1with a restriction enzyme into the appropriate sized fragments,subcloning the fragments into an appropriate cloning vector, and

conducting the dideoxy method using the subcloned fragments as atemplate. SEQ ID No. 34 of the Sequence Listing shows the nucleotidesequence of open reading frame present in the nucleotide sequence thusobtained. In addition, SEQ ID No. 35 of the Sequence Listing shows theamino acid sequence of the protease PFUS deduced from the nucleotidesequence.

Further, also when Bacillus subtilis DB104 transformed with the plasmidpSPT1, Bacillus subtilis DB104/pSPT1, is cultured, the protease activityis found in both a culture supernatant and an extract from the cells.SEQ ID No. 6 of the Sequence Listing shows the nucleotide sequence ofopen reading frame encoding a hybrid protein of the protease TCES andthe protease PFUS. In addition, SEQ ID No. 5 of the Sequence Listingshows the amino acid sequence of the hybrid protein deduced from thenucleotide sequence.

An amount of an expressed protease of the present invention can beincreased by utilizing a gene which is highly expressed in Bacillussubtilis, particularly a secretory protein gene. As such a gene, thegenes of α-amylase and the various extracellular proteases can be used.For example, an amount of the expressed protease PFUS can be increasedby utilizing the promoter and the signal sequence of subtilisin. Thatis, by ligating the full length protease PFUS gene to downstream of aregion encoding the signal sequence of subtilisin gene so that thetranslation frames of both genes coincide with each other, the proteasePFUS can be expressed as a fusion protein under the control ofsubtilisin gene promoter.

As the promoter and the signal sequence of subtilisin, those ofsubtilisin gene, which are inserted into the plasmid pKWZ, described inJ. Bacteriol., volume 171, page 2657-2665 (1989) can be used. Thenucleotide sequence of the gene is described in the above literature fora 5′ upstream region containing the promoter sequence and in J.Bacteriol., volume 158, page 411-418 (1984) for a region encodingsubtilisin, respectively. Based on these sequences, the primer SUB4 forintroducing the EcoRI site upstream of the promoter sequence of thegene, and the primer BmRI for introducing the BamHI site behind a regionencoding the signal sequence of subtilisin are synthesized,respectively. SEQ ID Nos. 36 and 37 of the Sequence Listing show thenucleotide sequences of the primers SUB4 and BmR1, respectively. Byusing the primers SUB4 and BmR1, an about 0.3 kb DNA fragment containingthe region encoding from the promoter to the signal sequence ofsubtilisin gene can be amplified by PCR using the plasmid pKWZ as atemplate.

The protease PFUS gene ligated downstream of the DNA fragment can betaken from a chromosomal DNA of Pyrococcus furiosus by the PCR method.As a primer which hybridizes with a 5′ part of the gene, the primerNPF-4 can be used. In addition, a primer which hybridizes with a 3′ partcan be made after the nucleotide sequence downstream of a terminationcodon of the gene is determined. That is, a portion of the nucleotidesequence of the plasmid pSNPD obtained by subcloning an about 0.6 kbfragment, produced by digestion of the plasmid pSNP1 with BamHI, intothe BamHI site of the plasmid vector pUC119 is determined (thenucleotide sequence is SEQ ID No. 38 of the Sequence Listing). Based onthe sequence, the primer NPM-1 which hybridizes with a 3′ part of theprotease PFUS gene and which can introduce the SphI site is synthesized.SEQ ID No. 39 of the Sequence Listing shows the sequence of the primerNPM-1.

On the other hand, when the protease PFUS gene is ligated to the above0.3 kb DNA fragment by utilizing the BamHI site, only one BamHI sitepresent in the gene becomes a barrier to the procedures. The primersmutRR and mutFR for removing this BamHI site by the PCR-mutagenesismethod can be made based on the nucleotide sequence of the protease PFUSgene shown in SEQ ID No. 34 of the Sequence Listing. The nucleotidesequences of the primers mutRR and mutRF are shown in SEQ ID Nos. 40 and41, respectively. In addition, when the BamHI site is removed byutilizing these primers, glycine present at the position 560 in theamino acid sequence of the protease PFUS shown in SEQ ID No. 35 of theSequence Listing is substituted with valine due to the nucleotidesubstitution which is introduced into the site.

By using these primers, the protease PFUS gene to be ligated to thepromoter to signal sequence-coding region of subtilisin gene can beobtained. That is, two kinds of PCRs are carried out using a chromosomalDNA of Pyrococcus furiosus as a template and using two kinds of pairs ofthe primers mutRR and NPF-4, and the primers mutFR and NPM-1. Further,the second PCR is carried out using a hetero duplex formed by mixing theDNA fragments amplified by both PCRs as a template, and using theprimers NPF-4 and NPM-1. Thus, the full length of the about 2.4 kbprotease PFUS gene containing no BamHI site can be amplified.

An about 2.4 kb DNA fragment obtained by digesting the abovePCR-amplified DNA fragment with BamHI and SphI is isolated, and replacedwith the BamHI-SphI fragment containing the protease PFUS gene in theplasmid pSNP1. The expression plasmid thus constructed was designatedpPS1 and Bacillus subtilis DB104 transformed with the plasmid wasdesignated Bacillus subtilis DB104/pPS1. When the transformant iscultured, the similar protease activity to that in a case of the use ofthe plasmid pSNP1 is found in both a culture supernatant and an extractfrom the cells, and it is confirmed that the substitution of the aminoacids dose not affect on the enzyme activity. FIG. 16 shows arestriction map of the plasmid pPS1.

An about 0.3 kb DNA fragment containing from the promoter to the signalsequence of the subtilisin is digested with EcoRI and BamHI, andsubstituted with the EcoRI-BamHI fragment containing the P43 promoterand the ribosome binding site in the plasmid pPS1. The expressionplasmid thus constructed was designated pNAPS1 and Bacillus subtilistransformed with the plasmid was designated Bacillus subtilisDB104/pNAPS1. The transformant is cultured, a culture supernatant and anextract from the cells are examined for the protease activity to befound that the protease activity is recognized in both samples. Anamount of expressed enzyme is increased as compared with Bacillussubtilis DB104/pSN1. FIG. 17 shows a restriction map of the plasmidpNAPS1.

By a similar method to that in a case of the protease TCES gene and theprotease PFUS gene, a protease gene having the homology with these genescan be obtained from hyperthermophiles other than Pyrococcus furiosusand Thermococcus celer. However, in PCR using the above oligonucleotidesPRO-1F, PRO-2F, PRO-2R and PRO-4R as a primer and using a chromosomalDNA of Staphylothermus marinus DSM3639 and that of Thermobacteroidesproteoliticus DSM5265 as a template, the amplification of a DNA fragmentas found in Thermococcus celer was not found.

In addition, it is known that the efficiency of gene amplification byPCR is largely influenced by the efficiency of annealing of a 3′terminal part of a primer and a template DNA. Even when theamplification of a DNA by PCR is not observed, a protease gene can bedetected by synthesizing and using the oligonucleotides having thedifferent nucleotide sequence from that used this time but encoding thesame amino acid sequence. Alternatively, a protease gene can be alsodetected by conducting southern hybridization using a chromosomal DNAand using the above oligonucleotides or a portion of otherhyperthermostable protease genes as a probe.

An about 1 kb DNA fragment encoding the sequence of residue 323 toresidue 650 of the amino acid sequence of the protease PFUL representedby SEQ ID No. 8 of the Sequence Listing is prepared, and this can beused as a probe to conduct genomic southern hybridization using achromosomal DNA of Staphylothermus marinus DSM3639 and that ofThermobacteroides proteoliticus DSM5265. As a result, when theStaphylothermus marinus chromosomal DNA digested with PstI (manufacturedby Takara Shuzo Co., Ltd.) is used, a signal is observed at the positionof about 4.8 kb. On the other hand, when the Thermobacteroidesproteoliticus chromosomal DNA digested with XbaI is used, a signal isobserved at the position of about 3.5 kb.

From this, it is revealed that a sequence having the homology with theprotease PFUL, the protease PFUS and the protease TCES gene is presentalso in the Staphylothermus marinus and Thermobacteroides proteoliticusDNA chromosomes. From the DNA fragment thus detected, a gene encoding ahyperthermostable protease present in Staphylothermus marinus orThermobacteroides proteoliticus can be isolated and identified by usingthe same method as that when the gene encoding the protease TCES or theprotease PFUS is isolated and identified.

The transformant in which the protease TCES gene, a hyperthermostableprotease gene of the present invention, is introduced (Bacillus subtilisDB104/pSTC3) expresses a hyperthermostable protease in a culture byculturing at 37° C. in LB medium containing 10 μg/ml kanamycin. Afterthe completion of cultivation, crude enzyme preparation is obtained bysubjecting centrifugation of a culture to collect a supernatant, andsalting out with ammonium sulfate and dialysis. Thus, the crude enzymepreparation obtained from Bacillus subtilis DB104/pSTC3 was designatedTC-3.

According to the similar procedures, a crude enzyme preparation can beobtained from the transformant Bacillus subtilis DB101/pSNP1 in whichthe protease PFUS gene is introduced, or from the transformant Bacillussubtilis DB104/pSPT1 in which a gene encoding a hybrid protease of theprotease TCES and the protease PFUS. Crude enzyme preparations obtainedfrom Bacillus subtilis DB104/pSNP1 and Bacillus subtilis DB104/pSPT1were designated NP-1 and PT-1, respectively.

Transformant Bacillus subtilis DB104/pNAPS1 in which the protease PFUSgene, a hyperthermostable protease gene of the present invention, isintroduced expresses a hyperthermostable protease in the cells orculture under the conventional conditions, for example, by culturing at37° C. in LB medium containing 10 μg/ml kanamycin. After the completionof cultivation, the cells and a culture supernatant are separated bycentrifugation, from either of which a crude enzyme preparation of theprotease PFUS can be obtained by the following procedures.

When an enzyme is purified from the cells, the cells are first lysed bythe lysozyme treatment, the lysate is heat-treated and centrifuged torecover a supernatant. This supernatant can be fractionated withammonium sulfate and subjected to hydrophobic chromatography to obtain apurified enzyme. The purified enzyme preparation thus obtained fromBacillus subtilis DB104/pNAPS1 was designated NAPS-1.

On the other hand, the culture supernatant is dialyzed and subjected toanion-exchange chromatography. The eluted active fractions can becollected, heat-treated, fractionated with ammonium sulfate, andsubjected to hydrophobic chromatography to obtain a purified enzyme ofthe protease PFUS. The purified enzyme preparation was designatedNAPS-1S.

When the purified products NAPS-1 and NAPS-1S thus obtained aresubjected to SDS-polyacrylamide gel electrophoresis, both enzymepreparation show a single band corresponding to a molecular weight ofabout 45 kDa. These two enzyme preparation are substantially the sameenzyme preparation which have been converted into a mature (active-type)enzyme by removing a pro sequence by heat-treatment during thepurification procedures.

The protease preparation produced by the transformants in which ahyperthermostable protease gene obtained by the present invention isintroduced, for example, TC-3, NP-1, PT-1, NAPS-1 and NAPS-LS have thefollowing enzymatic and physicochemical properties.

(1) Activity

The enzymes obtained in the present invention hydrolyze gelatin toproduce the short-chain polypeptides. In addition, the enzymes hydrolyzecasein to produce short-chain polypeptides.

In addition, the enzymes obtained in the present invention hydrolyzesuccinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine-4-methylcoumarin-7-amide(Suc-Leu-Leu-Val-Tyr-MCA; SEQ ID NO:43) to produce a fluorescentmaterial (7-amino-4-methylcoumarin).

Further, the enzymes obtained in the present invention hydrolyzesuccinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide(Suc-Ala-Ala-Pro-Phe-p-NA; SEQ ID NO:44) to produce a yellow material(p-nitroaniline).

(2) Method for Measuring Enzyme Activity

The enzyme activity of the enzyme preparations obtained in the presentinvention can be measured using a synthetic peptide substrate.

The enzyme activity of the enzyme preparation TC-3 obtained in thepresent invention can be measured using as a substrateSuc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO:43) (manufactured by PeptideLaboratory). That is, the enzyme preparation to be detected for theenzyme activity is appropriately diluted, to 20 μl of the solution isadded 80 μl of a 0.1M sodium phosphate buffer (pH 7.0) containing 62.5μM Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO:43), followed by incubating at 75°C. for 30 minutes. After the reaction is stopped by the addition of 20μl of 30% acetic acid, the fluorescent intensity is measured at theexcitation wavelength of 355 nm and the fluorescence wavelength of 460nm to quantitate an amount of the generated 7-amino-4-methylcoumarin,and the resulting value is compared with that obtained when incubatingwithout the addition of the enzyme preparation, to investigate theenzyme activity. The enzyme preparation TC-3 obtained by the presentinvention had the Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO:43) hydrolyzingactivity measured at pH 7.0 and 75° C.

In addition, the enzyme activity of the enzyme preparations NP-1, PT-1,NAPS-1 and NAPS-1S can be photometrically measured usingSuc-Ala-Ala-Pro-Phe-p-NA (SEQ ID NO:44) (manufactured by Sigma) as asubstrate. That is, an enzyme preparation to be detected for the enzymeactivity was appropriately diluted, to 50 μl of the solution was added50 μl of a 0.1M potassium phosphate buffer (pH 7.0) containingSuc-Ala-Ala-Pro-Phe-p-NA (SEQ ID NO:44) (Suc-Ala-Ala-Pro-Phe-p-NA(SEQ IDNO:44) solution), followed by incubating at 95° C. for 30 minutes. Afterthe reaction was stopped by ice-cooling, the absorbance at 405 nm wasmeasured to quantitate an amount of the generated p-nitroaniline, andthe resulting value was compared with that when incubating without theaddition of the enzyme preparation, to investigate the enzyme activity.Upon this, a 0.2 mM solution of Suc-Ala-Ala-Pro-Phe-p-NA (SEQ ID NO:44)was used for the enzyme preparations NP-1 and PT-1 and a 1 mM solutionwas used for the enzyme preparations NAPS-1 and NAPS-1S. The enzymepreparations NP-1, PT-1, NAPS-1 and NAPS-1S obtained by the presentinvention have the Suc-Ala-Ala-Pro-Phe-p-NA (SEQ ID NO:44) hydrolyzingactivity at measured pH 7.0 and 95° C.

(3) Detection of Activity on Various Substrates

The activity of the enzyme preparations obtained in the presentinvention on the synthetic peptide substrates is confirmed by a methodfor measuring the enzyme activity described in the above (2). That is,the enzyme preparation TC-3 obtained in the present invention has theSuc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO:43) hydrolyzing activity, and theenzyme preparations NP-1, PT-1, NAPS-1 and NAPS-1A have theSuc-Ala-Ala-Pro-Phe-p-NA (SEQ ID NO:44) hydrolyzing activity,respectively. In addition, the enzyme preparations NP-1, PT-1, NAPS-1and NAPS-1S were investigated for the Suc-Leu-Leu-Val-Tyr-MCA (SEQ IDNO:43) hydrolyzing activity by the enzyme activity measuring methoddescribed in the above (2) used for the enzyme preparation TC-3, and itwas shown that these enzyme preparations had the activity to degrade thesubstrates. Further, the enzyme preparation TC-3 was investigated forthe Suc-Ala-Ala-Pro-Phe-p-NA (SEQ ID NO:44) hydrolyzing activity by theenzyme activity measuring method described in the above (2) used for theenzyme preparations NP-1 and PT-1, and the activity to degrade thesubstrate was recognized. In addition, the activity of the enzymepreparations obtained in the present invention on gelatin can bedetected by confirming the degradation of gelatin by an enzyme on theSDS-polyacrylamide gel. That is, the enzyme preparation to be detectedfor the enzyme activity was appropriately diluted, to 10 μl of thesample solution was added 2.5 μl of a sample buffer (50 mM Tris-HCl, pH7.5, 5% SDS, 5% 2-mercaptoethanol, 0.005% Bromophenol Blue. 50%glycerol), followed by treatment at 100° C. for 5 minutes andelectrophoresis using 0.1% SDS-10% polyacrylamide gel containing 0.05%gelatin. After the completion of run, the gel was soaked in a 50 mMpotassium phosphate buffer (pH 7.0), and incubated at 95° C. for 3 hoursto carry out the enzyme reaction. Then, the gel was stained in 2.5%Coomassie Brilliant Blue R-250, 25% ethanol and 10% acetic acid for 30minutes, and transferred in 7% acetic acid to remove the excess dye over3 to 15 hours. The presence of the protease activity was detected by thefact that gelatin is hydrolyzed by a protease into peptides which arediffused out of the gel and, consequently, the relevant portion of thegel was not stained with Coomassie Brilliant Blue. The enzymepreparations TC-3, NP-1, PT-1, NAPS-1 and NAPS-LS obtained by thepresent invention had the gelatin hydrolyzing activity at 95° C.

In addition, the enzyme preparations NP-1, NAPS-1 and NSPA-1S derivedfrom the protease PFUS gene are recognized to have the gelatinhydrolyzing activity at the almost same positions on the gel in theabove activity measuring method. From this, it is shown that, in theseenzyme preparations, the processing from a precursor enzyme into amature type enzyme occurs in the similar manner.

Further, the hydrolyzing activity on casein can be detected according tothe same method as that used for detecting the activity on gelatinexcept that 0.1% SDS-10% polyacrylamide gel containing 0.05% casein isused. The enzyme preparations TC-3, NP-1, PT-1, NAPS-1 and NAPS-LSobtained by the present invention had the casein hydrolyzing activity at95° C.

Alternatively, the casein hydrolyzing activity of the enzymepreparations TC-3, NP-1, NAPS-1 and NAPS-1S obtained by the presentinvention can be measured by the following method. 100 μl of anappropriately diluted enzyme preparation was added to 100 μl of a 0.1Mpotassium phosphate buffer (pH 7.0) containing 0.2% casein, incubated at95° C. for 1 hour, and the reaction was stopped by the addition of 100μl of 15% trichloroacetic acid. An amount of an acid-soluble short-chainpolypeptide contained in the supernatant obtained by centrifugation ofthis reaction mixture was determined from the absorbance at 280 nm andcompared with that when incubating without the addition of an enzymepreparation, to investigate the enzyme activity. The enzyme preparationsTC-3, NP-1, NAPS-1 and NAPS-1S obtained by the present invention had thecasein hydrolyzing activity at 95° C.

(4) Optimum Temperature

The optimum temperature of the enzyme preparation TC-3 obtained by thepresent invention was investigated using the enzyme activity measuringmethod shown in the above (2) except for varying a temperature. As shownin FIG. 18, the enzyme preparation TC-3 showed the activity at atemperature of 37 to 95° C. and the optimum temperature thereof was 70to 80° C. That is, FIG. 18 is a figure showing the relationship betweenthe activity of the enzyme preparation TC-3 obtained in the presentinvention and a temperature, and the ordinate shows the relativeactivity to the maximum activity (%) and the abscissa shows atemperature.

In addition, the optimum temperature of the enzyme preparation NAPS-1obtained in the present invention was investigated by using the enzymeactivity measuring method shown in the above (2) except for varying atemperature. As shown in FIG. 19, the enzyme preparation NAPS-1 had theactivity at a temperature between 40 to 110° C. at the measuringconditions of pH 7.0, and the optimum temperature being 80 to 95° C.That is, FIG. 19 is a figure showing the relationship between theactivity of the enzyme preparation NAPS-1 obtained in the presentinvention and a temperature, and the ordinate shows the relativeactivity to the maximum activity (%) and the abscissa shows atemperature.

(5) Optimum pH

The optimum pH of the enzyme preparation TC-3 obtained by the presentinvention was investigated by the enzyme activity measuring method shownin the above (2). That is, the Suc-Leu-Leu-Val-Tyr-MCA (SEQ ID NO:44)solutions were prepared using the buffers having various pHs, and theenzyme activities obtained by using these solutions were compared. As abuffer, a sodium acetate buffer was used at pH 3 to 6, a sodiumphosphate buffer at pH 6 to 8, a sodium borate buffer at pH 8 to 9, anda sodium phosphate-sodium hydroxide buffer at pH 10 to 11. As shown inFIG. 20, the enzyme preparation TC-3 shows the activity at pH 5.5 to 9,and the optimum pH was pH 7 to 8. That is, FIG. 20 is a figure showingthe relationship between the activity of the enzyme preparation TC-3obtained in the present invention and pH, and the ordinate shows therelative activity (%) and the abscissa shows pH.

In addition, the optimum pH of the enzyme preparation NP-1 obtained inthe present invention was investigated by the enzyme activity measuringmethod shown in the above (2). That is, the Suc-Ala-Ala-Pro-Phe-pNA (SEQID NO:44) solutions were prepared by using the buffers having variouspHs, and the enzyme activities obtained by using these solution werecompared. As a buffer, a sodium acetate buffer was used at pH 4 to 6, apotassium phosphate at pH 6 to 8, a sodium borate buffer at pH 5 to 10,and a sodium phosphate-sodium hydroxide buffer at pH 10.5. As shown inFIG. 21, the enzyme preparation NP-1 shows the activity at pH 5 to 10,and the optimum pH was pH 5.5 to 8. That is, FIG. 21 is a figure showingthe relationship between the activity of the enzyme preparation NP-1obtained in the present invention and pH, and the ordinate shows therelative activity (%) and the abscissa shows pH.

Further, the optimum pH of the enzyme preparation NAPS-1 obtained in thepresent invention was investigated by the enzyme activity measuringmethod shown in the above (2). That is, the Suc-Ala-Ala-Pro-Phe-pNA (SEQID NO:44) solutions were prepared by using the buffers having variouspHs, and the enzyme activities obtained by using these solution werecompared. As a buffer, a sodium acetate buffer was used at pH 4 to 6, apotassium phosphate at pH 6 to 8, a sodium borate buffer at pH 8.5 to10. As shown in FIG. 22, the enzyme preparation NAPS-1 shows theactivity at pH 5 to 10, and the optimum pH was pH 6 to 8. That is, FIG.22 is a figure showing the relationship between the activity of theenzyme preparation NAPS-1 obtained in the present invention and pH, andthe ordinate shows the relative activity (%) and the abscissa shows pH.

(6) Thermostability

The thermostability of the enzyme preparation TC-3 obtained by thepresent invention was investigated. That is, the enzyme preparation wasincubated at 80° C. in 20 mM Tris-HCl, pH 7.5 for various periods oftime, an appropriate amount thereof was taken to measure the enzymeactivity by the method shown in the above (2), and the activity wascompared with that when not heat-treated. As shown in FIG. 23, theenzyme preparation TC-3 obtained by the present invention had not lessthan 90% of the activity even after the heat-treatment for 3 hours and,thus, was stable on the above heat-treatment. That is, FIG. 23 is afigure showing the thermostability of the enzyme preparation TC-3obtained in the present invention, and the ordinate shows the residualactivity (%) after the heat-treatment and the abscissa shows time.

In addition, the thermostability of the enzyme preparation NP-1 obtainedin the present invention was investigated. That is, the enzymepreparation was incubated at 95° C. in 20 mM Tris-HCl, pH 7.5 forvarious periods of time, an appropriate aliquot was taken to determinethe enzyme activity by the method shown in the above (2), and the enzymeactivity was compared with that when not heat-treated. As shown in FIG.24, the enzyme preparation NP-1 obtained in the present invention isobserved to have the remarkably increased enzyme activity when incubatedat 95° C. This is considered to be because a protease produced as aprecursor causes the self-catalytic activation during theheat-treatment. In addition, no decrease in the activity was recognizedin the heat-treatment for up to 3 hours. That is, FIG. 24 is a figureshowing the thermostability of the enzyme preparation NP-1 obtained inthe present invention, and the ordinate shows the residual activity (%)after the heat-treatment and the abscissa shows time.

In addition, the above enzyme preparation NP-1 activated by theheat-treatment was investigated for the thermostability. That is, theenzyme preparation NP-1 was activated by the heat-treatment at 95° C.for 30 minutes, incubated at 95° C. for various periods of time, and theactivity was determined as described above to compare with that when notheat-treated. At the same time, buffers having the various pHs (sodiumacetate buffer at pH 5, potassium phosphate buffer at pH 7, sodiumborate buffer at pH9, sodium phosphate-sodium hydroxide buffer at pH 11,20 mM in every case) were used. As shown in FIG. 25, when the activatedenzyme preparation NP-1 obtained in the present invention was treated ina buffer at pH 9, it had not less than 90% of the activity after theheat-treatment for 8 hours and approximately 50% of the activity evenafter the heat-treatment for 24 hours and, thus, being very stable tothe above heat-treatment. That is, FIG. 25 is a figure showing thethermostability of the enzyme preparation NP-1 obtained in the presentinvention, and the ordinate shows the residual activity (%) after theheat-treatment and the abscissa shows time.

In addition, the enzyme preparation NAPS-1 obtained by the presentinvention was investigated for the thermostability. That is, atemperature of the enzyme preparation was maintained at 95° C. in 20 mMTris-HCl, pH 7.5 for various periods of time, an appropriate aliquot wastaken to determine the enzyme activity by the method shown in the above(2) to compare with that when not heat-treated. As shown in FIG. 26, theenzyme preparation NAPS-1 obtained by the present invention had not lessthan 80% of the activity even after the heat-treatment at 95° C. for 3hours and, thus, being stable against the above heat-treatment. That is,FIG. 26 is a figure showing the thermostability of the enzymepreparation NAPS obtained in the present invention, and the ordinateshows the residual activity (%) after the heat-treatment and theabscissa shows time.

(7) pH Stability

The pH stability of the enzyme preparation NP-1 obtained by the presentinvention was investigated

according to the following procedures. Each 50 μl of 20 mM buffers atvarious pHs, which contain the enzyme preparation NP-1 activated by theheat-treatment at 95° C. for 30 minutes, was treated at 95° C. for 60minutes, and an appropriate aliquot was taken to determine the enzymeactivity by the method shown in the above (2) to compare with that whennot treated. As a buffer, a sodium acetate buffer was used at pH 4 to 6,a potassium phosphate buffer at pH 6 to 8, a sodium borate buffer at pH9 to 10, a sodium phosphate-sodium hydroxide buffer at pH 11. As shownin FIG. 27, the enzyme preparation NP-1 obtained by the presentinvention retained not less than 95% of the activity even after thetreatment at 95° C. for 60 minutes at pH between 5 and 11. That is, FIG.27 is a figure showing the pH stability of the enzyme obtained by thepresent invention, and the ordinate shows the residual activity (%) andabscissa shows pH.

(8) Stability to Detergent

The stability to detergent of the enzyme preparation NP-1 obtained bythe present invention was investigated using SDS as detergent. Theenzyme preparation NP-1 was activated by the heat-treatment at 95° C.for 30 minutes. Each 50 μl of a solution containing only the enzymepreparation and a solution further

containing SDS to the final concentration of 0.1% or 1% was prepared.These solutions were incubated at 95° C. for various periods of time, anappropriate amount thereof was taken to determine the enzyme activity bythe method described in the above (2) to compare with that when nottreated. As shown in FIG. 28, the activated enzyme preparation NP-1obtained by the present invention had not less than 80% of the activityafter the heat-treatment at 95° C. for 8 hours and approximately 50% ofthe activity even the after heat-treatment for 24 hours independently ofthe presence of SDS and, thus, having the high stability even in thepresence of SDS. That is, FIG. 28 is a figure showing the stability toSDS of the enzyme preparation NP-1 obtained by the present invention,and the ordinate shows the residual activity (%) and the abscissa showstime.

In addition, the stability to detergent of the enzyme preparation NAPS-1obtained by the present invention was investigated using SDS asdetergent. Each 50 μl of a solution containing only the enzymepreparation NAPS-1 and a solution further containing SDS to the finalconcentration of 0.1% or 1% was prepared. These solutions was incubatedat 95° C. for various periods of time, an appropriate aliquot was takento determine the enzyme activity by the method described in the above(2) to compare with that when not treated. As shown in FIG. 29, theenzyme preparation NAPS-1 obtained by the present invention hadapproximately 80% of the activity after the heat-treatment at 95° C. for3 hours independently of the presence of SDS. That is, FIG. 29 is afigure showing the stability to SDS of the activated enzyme preparationNAPS-1 obtained by the present invention, and the ordinate shows theresidual activity (%) and the abscissa shows time.

When the above results are compared, it is shown that the enzymepreparation NAPS-1 has remarkably decreased residual activity incomparison with the enzyme preparation NP-1. However, this phenomenon ishardly considered to be based on the difference in the stability to SDSof the enzyme proteins themselves contained in both preparations. It isthought to be the cause for the above phenomenon that NAPS-1 which isthe purified enzyme preparation has less contaminant proteins ascompared with NP-1 and, thereby, the inactivation easily occurs due toself-digestion.

(9) Stability to Organic Solvent

The stability to an organic solvent of the enzyme preparation NAPS-1obtained by the present invention was investigated using acetonitrile.Each 50 μl of enzyme preparation NAPS-1 solutions containingacetonitrile to the final concentration of 25% or 50% was incubated at95° C. for various periods of time, and an appropriate aliquot was takento determine the activity by the method described in the above (2) tocompare with that when not treated. As shown in FIG. 30, the enzymepreparation NAPS-1 obtained by the present invention had the activity ofnot less than 80% of that before the treatment, even after the treatmentat 95° C. for 1 hour in the presence of 50% acetonitrile. That is, FIG.30 is a figure showing the stability to acetonitrile of the enzymepreparation NAPS-1 obtained by the present invention.

(10) Stability to Denaturing Agent

The stability to various denaturing agents of the enzyme preparationNAPS-1 obtained by the present invention was investigated using urea andguanidine hydrochloride. Each 50 μl of the enzyme preparation NAPS-1solution containing urea to the final concentration of 3.2 M or 6.4 M orguanidine hydrochloride to the final concentration of 1 M, 3.2 M or 6.4M was prepared. These solutions were incubated at 95° C. for variousperiods of time, an appropriate aliquot was taken to determine theactivity by the method described in the above (2) to compare with thatwhen not treated. As shown FIG. 31, the enzyme preparation NAPS-1obtained by the present invention shows the

resistance to urea and had the activity of not less than 70% of thatbefore the treatment, even after the treatment at 95° C. for 1 hour inthe presence of 6.4 M urea. That is, FIG. 31 is a figure showing thestability to urea and FIG. 32 is a figure showing the stability toguanidine hydrochloride, and the ordinate indicates the residualactivity and the abscissa indicates time.

(11) Effects of Various Reagents

The effects of various reagents on the enzyme preparations TCES andNAPS-1 obtained by the present invention were investigated. That is, theabove enzyme preparations were treated at 37° C. for 30 minutes in thepresence of the various reagents at the final concentration of 1 mM, andan aliquot thereof was taken to determine the enzyme activity by themethod described in the above (2) to compare with that (control) when noreagent was added. The results are shown in Table 1.

TABLE 1 Reagent TCES NAPS-1 Control   100%   100% EDTA 103.5%  36.1%PMSF  8.1%  0.1% Antipain  19.0%  81.9% Chymostatin    0%  6.6%Leupeptin 104.5%  89.3% Pepstatin 105.2% 100.7% N-ethylmaleimide  82.6%102.6%

As shown in Table 1, when treated with PMSF (phenylmethanesulfonylfluoride) and chymostatin, both enzyme preparations had the remarkablydecreased activity. In addition, when treated with antipain, thedecrease in the activity was observed in TCES, and when treated withEDTA, in NAPS-1, respectively. In a case of other reagents, the largedecrease was not observed in the activity.

(12) Molecular Weight

A molecular weight of the enzyme preparation NAPS-1 obtained by thepresent invention was determined by SDS-PAGE using 0.1% SDS-10%polyacrylamide gel. The enzyme preparation NAPS-1 showed a molecularweight of about 45 kDa on SDS-PAGE. On the other hand, the enzymepreparation NAPS-1S showed the same molecular weight as that of theenzyme preparation NAPS-1.

(13) N-Terminal Amino Acid Sequence

The N-terminal amino acid sequence of a mature enzyme, the proteasePFUS, was determined using the enzyme preparation NAPS-1 obtained by thepresent invention. The enzyme preparation NAPS-1 electrophoresed on 0.1%SDS-10% polyacrylamide gel was transferred onto the PVDF membrane, andthe N-terminal amino acid sequence of the enzyme on the membrane wasdetermined by the automated Edman degradation using a protein sequencer.The N-terminal amino acid sequence of the mature type protease PFUS thusdetermined is shown in SEQ ID No. 42 of the Sequence Listing. Thesequence coincided with the sequence of amino acids 133 to 144 in theamino acid sequence of the protease PFUS represented by SEQ ID No. 35 ofthe Sequence Listing, and it was shown that the mature protease PFUS isan enzyme consisting of the polypeptides including behind this part. Theamino acid sequence of the mature protease PFUS thus revealed isrepresented by SEQ ID No. 3 of the Sequence Listing. In addition, asdescribed above, there is no influence on the enzyme activity of theprotease PFUS independently of whether 428th amino acid (correspondingto 560th amino acid in the amino acid sequence represented by SEQ ID No.35 of the Sequence Listing) is glycine or valine. Further, within thenucleotide sequence of the protease PUFS gene represented by SEQ ID No.34 of the Sequence Listing, that of a region encoding the mature typeenzyme is shown in SEQ ID No. 4. 1283rd base in the sequence may beguanine or thimine.

In a case of in vitro gene amplification by PCR, the misincorporation ofa nucleotide may occur during the elongation reaction, leading to thenucleotide substitution in the sequence of the resulting DNA. Thisfrequency largely depends upon the kind of the enzyme used for PCR, thecomposition of the reaction mixture, the reaction conditions, thenucleotide sequence of a DNA to be amplified and the like. However, whena certain region in a gene is simply amplified as performed usually, thefrequency is at best around one nucleotide per 400 nucleotides. In thepresent invention, PCR was used for isolation of a gene of the proteaseTCES or the protease PFUS or construction of the expression plasmidtherefor. The number of nucleotide substitutions in the nucleotidesequence of the resulting gene is, if any, a few nucleotides. Takinginto consideration the fact that the nucleotide substitution on a genedose not necessarily lead to the amino acid substitution in theexpressed protein due to degeneracy of translation codons, the number ofthe possible amino acid substitutions can be evaluated to be at best 2to 3 in the whole residues. It cannot be denied that the nucleotidesequence of a gene of the protease TCES and the protease PFUS and theamino acid sequence of the proteases disclosed herein are different fromnatural ones. However, the object of the present invention is todisclose a hyperthermostable protease having the high activity at hightemperature and a gene encoding the same and, therefore, the proteaseand the gene are not limited to the same enzyme and the same geneencoding the same as the natural ones. And it is clear to those skilledin the art that even a gene having the possible nucleotide substitutioncan hybridize to a natural gene under the stringent conditions.

Further, in the specification, a method for obtaining a gene of interestis clearly disclosed such that (1) the library for expression cloning ismade from a chromosomal DNA of the hyperthermophiles and the expressionof the protease activity is screened, (2) a gene possibly expressing thehyperthermostable protease is isolated by hybridization or PCR based onthe homology of amino acid sequences, and the enzyme action ofexpression products of these genes, that is, the hyperthermostableprotease activity is confirmed using an appropriate microorganism.Therefore, it can be easily determined by using the above method whetherthe gene sequence with the mutation introduced encodes ahyperthermostable protease, after a variety of mutations are introducedinto the hyperthermostable protease gene of the present invention usingthe known mutation introducing method. The kind of the mutation to beintroduced is not limited to specified ones as long as the gene sequenceobtained as a result of the mutation introduction expressessubstantially the same protease activity as that of thehyperthermostable protease of the present invention. However, in orderthat the expressed protein retains the protease activity, the mutationis desirably introduced into a region other than four regions which areconserved in common in the serine proteases.

A mutation can be randomly introduced into any region of a gene encodingthe hyperthermostable protease (random mutagenesis), or alternatively, adesired mutation can be introduced into a specified pre-determinedposition (site-directed mutagenesis). As a method for randomlyintroducing a mutation, for example, there is a method for chemicallytreating a DNA. In this case, a plasmid is prepared such that a regioninto which a mutation is sought to be introduced is partiallysingle-stranded, and sodium bisulfite is acted on this partiallysingle-stranded region to convert a base cytosine into uracil and, thus,introducing a transition mutation from C:G to T:A. In addition, a methodfor producing a base substitution during a process where asingle-stranded part is repaired to double-strand in the presence of[α-S] dNTP is also known. The details of these methods are described inProc. Natl. Acad. Sci. USA, volume 79, page 1408-1412 (1982) and Gene,volume 64, page 313-319 (1988).

Random mutation can also be introduced by conducting PCR under theconditions where fidelity of the nucleotide incorporation becomes lower.In particular, the addition of manganese to the reaction system iseffective and the details of this method are described in Anal.Biochem., volume 224, page 347-355 (1995). As a method for introducing asite-directed mutation, for example, there is a method using a systemwhere a gene of interest is made single-stranded, a primer designeddepending upon a mutation sought to be introduced in thissingle-stranded part is synthesized, and the primer is annealed to thepart, which is introduced into in vivo system where only the strand witha mutation introduced is selectively replicated. The details of thismethod are described in Methods in Enzymology, volume 154, page 367(1987). For example, a mutation introducing kit, Mutant K manufacturedby Takara Shuzo Co., Ltd. can be used. Site-directed mutagenesis can beconducted also by PCR and the details are described of the method in PCRTechnology, page 61-70 (1989), edited by Ehlich and published by TakaraShuzo Co., Ltd. Alternatively, for example, LA-PCR in vitro mutagenesiskit manufactured by Takara Shuzo Co., Ltd. can be used. By using theabove method, a mutation of substitution, deletion and insertion can beintroduced.

Thus, an enzyme having the similar thermostability and optimumtemperature to those of the hyperthermostable protease of the presentinvention but having a little different, for example, optimum pH can beproduced in a host by introducing a mutation using as a base the

hyperthermostable protease gene of the present invention. In this case,the base nucleotide sequence of the hyperthermostable protease gene isnot necessarily limited to the sequence derived from onehyperthermostable protease.

A hybrid gene can be made by recombinating two or more hyperthermostableprotease genes having a sequence homologous to each other, such as thosedisclosed by the present invention, by exchanging the homologoussequence, and the hybrid enzyme encoded by the gene can be produced in ahost. Also in a case of a hybrid gene, whether it is a hyperthermostableprotease gene can be determined by testing for the enzyme action of thegene product, that is, the protease activity. For example, by using theabove plasmid pSPT1, a hybrid protease of which N-terminal part isderived from the protease PFUS and of which C-terminal part is derivedfrom the protease TCES can be produced in Bacillus subtilis, and thishybrid protease has the protease activity at 95° C.

The hybrid enzyme is expected to have the properties of two or more baseenzymes at the same time. For example, when the protease TCES and theprotease PFUS disclosed herein are compared, the protease TCES issuperior in respect of the extracellular secretion

efficiency and the protease PFUS is superior in respect of thethermostability. Since a signal sequence located at a N-terminal of theproteins has the great influence on extracellular secretion efficiency,if an expression plasmid is constructed so that a protein having, incontrast with pSPT1, a N-terminal part derived from the protease TCESand a C-terminal part derived from the protease PFUS is produced, ahyperthermostable protease having the equal thermostability to that ofthe protease PFUS can be secreted at the equal secretional efficiency tothat of the protease TCES. In addition, since a signal sequence is cutfrom an enzyme when the enzyme isextracellularly secreted, it has little influence on the nature of theenzyme itself. Therefore, when a hyperthermostable protease is producedusing a mesophile, its signal sequence dose not necessarily need to bederived from hyperthermophiles and a signal sequence derived from amesophile has no problem as long as a protein of interest isextracellularly secreted at a higher efficiency.

In particular, when a signal sequence of a secretory protein which ishighly expressed in a host to be used is employed, a higher secretion isexpected.

Upon construction of the above hybrid gene, a recombination dose notnecessarily need to be conducted site-directedly. Alternatively, ahybrid gene can be made, for example, by mixing two or more DNAs of ahyperthermostable protease gene, which are raw materials forconstruction of the hybrid gene, fragmenting these with a DNA degradingenzyme and reconstituting these fragments using a DNA polymerase. Thedetails of this method are described in Proc. Natl. Acad. Sci. USA,volume 91, page 10747-10751 (1994). Also in this case, a sequence of agene encoding a hyperthermostable gene can be isolated and identifiedfrom the resulting hybrid genes by examining the hyperthermostableprotease activity of expressed proteins as described above. In addition,it is expected that sequences encoding four regions common to the serineproteases are conserved in the sequences of the genes thus obtained.

Therefore, it is clear to those skilled in the art that the resultinghybrid gene can hybridize to a DNA selected from the oligonucleotidesPRO-1F, PRO-2F, PRO-2R and PRO-4R having the nucleotide sequencesrepresented by SEQ ID Nos. 9, 10, 11 and 12 of the Sequence Listing bythe appropriate hybridization conditions. In addition, it is also clearthat a novel hyperthermostable protease gene obtained by the abovemutation introduction can hybridize to a gene having a DNA sequenceselected from nucleotide sequences represented by SEQ ID Nos. 9, 10, 11and 12 of the Sequence Listing, for example, the protease PFUL gene bythe appropriate hybridization conditions.

In the specification, we described by focusing on obtaining of ahyperthermostable gene. However, a gene encoding a novel protease havingboth high thermostability and other properties can be made byconstructing a hybrid gene of the hyperthermostable protease gene of thepresent invention and a protease gene having a sequence homology withthe hyperthermostable protease gene of the present invention but havingno thermostability, for example, by constructing a hybrid gene with agene of subtilisin to improve the thermostability of subtilisin, toobtain a gene encoding a protease having the properties originallyretained by subtilisin and the higher thermostability.

In the present invention, we used Escherichia coli and Bacillus subtilisas a host into which a gene is introduced in order to detect theprotease activity retained by a protein encoded by a gene and produce anenzyme preparation. However, hosts into which a gene is introduced arenot limited to specified ones. Any hosts can be used as long as atransforming method is established for the hosts, such as Bacillusbrevis, Lactobacillus, yeast, mold fungi, animal cells, plant cells,insect cells and the like. Upon this, it is important that a polypeptideis folded such that an expressed protein becomes an active form and thisdoes not result in the harmful or lethal effect. Among hosts listedabove, Bacillus brevis, Lactobacillus and mold fungi which are known tosecret their products in a medium can be used as a host for massproduction of a protease of interest on an industrial scale, in additionto Bacillus subtilis.

EXAMPLES

The following Examples further describe the present invention in detailbut are not limit the scope thereof.

Example 1

(1) Preparation of Oligonucleotide for Detection of HyperthermostableProtease Gene

By comparing the amino sequence of the protease PFUL represented by SEQID No. 8 of the Sequence Listing with those of alkaline serine proteasesderived from the known bacterium, the homologous amino acid sequencescommon to them proved to exist. Among them, three regions were selectedand the oligonucleotides were designed, which were used as primers forPCR to detect hyperthermostable protease genes.

FIGS. 2, 3 and 4 show the relationship among the amino acid sequencescorresponding to the above three regions of the protease PFUL, thenucleotide sequences of the protease PFUL gene encoding the regions, andthe nucleotide sequences of the oligonucleotides PRO-1F, PRO-2F, PRO-2Rand PRO-4R synthesized based thereon. SEQ ID Nos. 9, 10, 11 and 12 showthe nucleotide sequences of the oligonucleotides PRO-1F, PRO-2F, PRO-2Rand PRO-4R, respectively.

(2) Preparation of Chromosomal DNA of Thermococcus celer

10 ml of a culture of Thermococcus celer DSM2476 obtained from DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH was centrifuged tocollect the cells which were suspended in 100 μl of 50 mM Tris-HCl, pH8.0 containing 25% sucrose. To this suspension was added 20 μl of 0.5 MEDTA and 10 μl of 10 mg/ml lysozyme, and was incubated at 20° C. for 1hour, 800 μl of a SET solution (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl,pH 8.0), 50 μl of 10% SDS and 10 μl of 20 mg/ml proteinase K were addedthereto, and was incubated at 37° C. for 1 hour. The reaction wasstopped by extraction with phenol-chloroform and precipitated withethanol to recover a DNA which was dissolved in 50 μl of a TE buffer (10mM Tris-HCl, pH 8.0, 0.1 mM EDTA) to give a chromosomal DNA solution.

(3) Detection of Hyperthermostable Protease Gene by PCR

A PCR reaction mixture was prepared from the above chromosomal DNA ofThermococcus celer and the oligonucleotides PRO-1F and PRO-2R, or PRO-2Fand PRO-4R, and a 35 cycles reaction was carried out, each cycleconsisting of 94° C. for 1 minute-55° C. for 1 minute-72° C. for 1minute. When an aliquot of these reaction mixture were subjected toagarose gel electrophoresis, amplification of three DNA fragments incase of the using the oligonucleotides PRO-1F and PRO-2R, and one DNAfragments in case of the using the oligonucleotides PRO-2F and PRO-4Rwere observed. These amplified fragments were recovered from the agarosegel, and the DNA ends thereof were made blunt using a DNA blunting kit(manufactured by Takara Shuzo Co., Ltd.) and phosphorylated using the T4polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.). Then,the plasmid vector pUC19 (manufactured by Takara Shuzo Co., Ltd.) wasdigested with HincII

(manufactured by Takara Shuzo Co., Ltd.), the resulting fragments weredephosphorylated at ends thereof by alkaline phosphatase (manufacturedby Takara Shuzo Co. Ltd.), mixed with the above PCR-amplified DNAfragments to allow to ligate, followed by introduction into Escherichiacoli JM109. Plasmids were prepared from the resultingtransformant, and the plasmids with an appropriate size DNA fragmentinserted were selected, followed by sequencing of the inserted fragmentby the dideoxy method.

Of these plasmids, the amino acid sequence deduced from the nucleotidesequence of the plasmid p1F-2R(2) containing an about 150 bp DNAfragment amplified using the oligonucleotides PRO-1F and PRO-2R, andthat deduced from the nucleotide sequence of the plasmid p2F-4Rcontaining an about 550 bp DNA fragment amplified using oligonucleotidesPRO-2F and PRO-4R contained sequences having the homology with the aminoacid sequences of the protease PFUL, subtilisin and the like. SEQ ID No.13 of the Sequence Listing shows the nucleotide sequence of the insertedDNA fragment in the plasmid p1F-2R(2) and the amino acid sequencededuced therefrom and SEQ ID NO. 14 of the Sequence Listing shows thenucleotide sequence of the inserted DNA fragment in the plasmid p2F-4Rand the amino acid sequence deduced therefrom. In the nucleotidesequence represented by SEQ ID No. 13 of the Sequence Listing, thesequence of 1st to 21st nucleotides and that of 113rd to 145thnucleotides and, in the nucleotide sequence represented by SEQ ID No. 14of the Sequence Listing, the sequence of 1st to 32nd nucleotides andthat of 532nd to 564th nucleotides are the sequences of theoligonucleotides (corresponding to oligonucleotides PRO-1F, PRO-2R,PRO-2F and PRO-4R, respectively) used as primers for PCR.

FIG. 5 shows a figure of a restriction map of the plasmid p2F-4R.

(4) Screening of Protease Gene Derived from Thermococcus celer

The chromosomal DNA of Thermococcus celer was partially digested withthe restriction enzyme Sau3AI (manufactured by Takara Shuzo Co., Ltd.),followed by partial repair of the DNA ends using Klenow Fragment(manufactured by Takara Shuzo Co., Ltd.) in the presence of dATP anddGTP. The DNA fragments were mixed with the lambda GEM-11 XhoI Half-SiteArms Vector (manufactured by Promega) to allow to ligate, which wassubjected in vitro packaging using Gigapack Gold (manufactured byStratagene) to prepare a lambda phage library containing the chromosomalDNA fragments of Thermococcus celer. A part of the library wastransformed into Escherichia coli LE392 (manufactured by Promega) toform the plaques on a plate, and the plaques were transferred toHybond-N+membrane (manufactured by Amersham). After transference, themembrane was treated with 0.5N NaOH containing 1.5M NaCl, then with 0.5MTris-HCl, pH 7.5 containing 3M NaCl, washed with 6×SSC, air dried, andirradiated with ultraviolet rays on the UV transilluminator to fix thephage DNA to the membrane.

On the other hand, the plasmid p2F-4R was digested with PmaCI and StuI(both manufactured by Takara Shuzo Co., Ltd.), which was subjected to 1%agarose gel electrophoresis to recover the separated about 0.5 kb DNAfragment. By using this fragment as a template and using Random PrimerDNA Labeling Kit Ver. 2 (manufactured by Takara Shuzo Co., Ltd.) and[α-³²P]dCTP (manufactured by Amersham), a ³²P-labeled DNA probe wasprepared.

The membrane with the DNA fixed thereto was treated with a hybridizationbuffer (6×SSC containing 0.5% SDS, 0.1% SBA, 0.1% polyvinylpyrrolidone,0.1% Ficoll 400, 0.01% denatured salmon sperm DNA) at 50° C. for 2hours, and transferred to the same buffer containing the ³²P-labeled DNAprobe, followed by hybridization at 50° C. for 15 hours. After thecompletion of hybridization, the membrane was washed with 2×SSCcontaining 0.5% SDS at room temperature, then with 1×SSC containing 0.5%SDS at 50° C. The membrane was further rinsed with 1×SSC, air dried anda X-ray film was exposed thereto at −80° C. for 6 hours to obtain anautoradiogram. About 3,000 phage clones were screened and, as a result,one clone containing a protease gene was obtained. Based on the signalon the autoradiogram, the position of this phage clone was found and theplaque corresponding on the plate used for transfer to the membrane wasisolated into 1 ml of a SM buffer (50 mM Tris-HCl, pH 7.5, 1M NaCl, 8 mMMgSO4, 0.01% gelatin) containing 1% chloroform.

(5) Detection of Phage DNA Fragment Containing Protease Gene Derivedfrom Thermococcus celer

Transduced Escherichia coli LE392 using the above phage clone wascultured in the NZCMY medium (manufactured by Bio101) at 37° C. for 15hours to obtain a culture, from which a supernatant was collected toprepare a phage DNA using QIAGEN-lambda kit (manufactured by QIAGEN).The resulting phage DNAs were digested with BamHI, EcoRI, EcoRV, HincII,KpnI, NcoI, PstI, SacI, Sal1, SmaI and SphI (all manufactured by TakaraShuzo Co., Ltd.), respectively, followed by agarose gel electrophoresis.Then, DNAs were transferred from the gel to Hybond-N+ membrane accordingto the southern transfer method described in Molecular Cloning; ALaboratory Manual, 2nd edition (1986), edited by T. Maniatis, et al.,published by Cold Spring Harbor Laboratory.

The resulting membrane was treated in a hybridization buffer at 50° C.for 4 hours, and transferred to the same buffer containing the³²P-labeled DNA probe used in Example 1-(4), followed by hybridizationat 50° C. for 18 hours. After the completion of hybridization, themembrane was washed in 1×SSC containing 0.5% SDS at 50° C., then rinsedwith 1×SSC and air dried. The membrane was exposed to a X-ray film at−80° C. for 6 hours to obtain an autoradiogram. This autoradiogramindicated that an about 9 kb DNA fragment contained a protease gene incase of the phage DNA digested with KpnI.

Then, the phage DNA containing the above protease gene was digested withKpnI, and further digested successively with BamHI, PstI and SphI,followed by 1% agarose gel electrophoresis. According to the similarprocedures to those described above, southern hybridization wasconducted and it was indicated that an about 5 kb KpnI-BamHI fragmentcontained a protease gene.

(6) Cloning of DNA Fragment Containing Protease Gene Derived fromThermococcus celer

The phage DNA containing the above protease gene was digested with KpnIand BamHI, which was subjected to 1% agarose gel electrophoresis toseparate and isolate an about 5 kb DNA fragment from the gel. Then, theplasmid vector pUC119 (manufactured by Takara Shuzo Co., Ltd.) wasdigested with KpnI and BamHI, which was mixed with the above about 5 kbDNA fragment to allow to ligate, followed by introduction intoEscherichia coli JM109. Plasmids were prepared form the resultingtransformant, the plasmid containing the about 5 kb DNA fragment wasselected and designated the plasmid pTC3.

FIG. 6 shows a restriction map of the plasmid pTC3.

(7) Preparation of Plasmid pTCS6 Containing Protease Gene Derived fromThermococcus celer

The above plasmid pTC3 was digested with SacI, which was electrophoresedusing 1% agarose gel, and southern hybridization was carried outaccording to the same manner as that described in Example 1-(5) for

detecting the phage DNA fragment containing a protease gene. A signal onthe resulting autoradiogram indicated that an about 1.9 kb DNA fragmentobtained by digesting the plasmid pTC3 with SacI contained ahyperthermostable protease gene.

Then, the plasmid pTC3 was digested with SacI, which was subjected to 1%agarose gel electrophoresis to isolate an about 1.9 kb DNA fragment.Then, the plasmid vector pUC118 (manufactured by Takara Shuzo Co., Ltd.)was digested with SacI, which was dephosphorylated using alkalinephosphatase and mixed with the about 1.9 kb fragment to allow to ligate,followed by introduction into Escherichia coli JM109. Plasmids wereprepared from the resulting transformant, and the plasmid containingonly one molecule of the about 1.9 kb fragment was selected anddesignated the plasmid pTCS6.

FIG. 7 shows a restriction map of the plasmid pTCS6.

(8) Determination of Nucleotide Sequence of DNA Fragment Derived fromThermococcus celer Contained in Plasmid pTCS6

In order to determine the nucleotide sequence of the protease genederived from Thermococcus celer inserted into the plasmid pTCS6, thedeletion mutants wherein the DNA fragment portion inserted into theplasmid had been deleted in various length were prepared using KiloSequence Deletion Kit (manufactured by Takara Shuzo Co., Ltd.). Amongthem, several mutants having suitable length of deletion were selectedand the nucleotide sequence of each of the inserted DNA fragment partswas determined by the dideoxy method, and these results were combined to

determine the nucleotide sequence of the inserted DNA fragment containedin the plasmid pTCS6. SEQ ID No. 15 of the Sequence Listing shows theresulting nucleotide sequence.

(9) Cloning of 5′ Upstream Region of a Protease Gene Derived fromThermococcus celer by PCR Using Cassette and Cassette Primer

A 5′ upstream region of the protease gene derived from Thermococcusceler was obtained by using LA PCR in vitro cloning kit (manufactured byTakara Shuzo Co., Ltd.).

Based on the nucleotide sequence of the inserted DNA fragment containedin the plasmid pTCS6 represented by SEQ ID No. 15 of the SequenceListing, the primer TCE6R for use in cassette PCR was synthesized. SEQID No. 16 of the Sequence Listing shows the nucleotide sequence of theprimer TCE6R.

Then, a chromosomal DNA of Thermococcus celer was completely digestedwith HindIII (manufactured by Takara Shuzo Co., Ltd.), and the fragmentswere ligated to the HindIII cassette (manufactured by Takara Shuzo Co.,Ltd.) by the ligation reaction. By using this as a template, a PCRreaction mixture containing the primer TCE6R and the cassette primer C1(manufactured by Takara Shuzo Co., Ltd.) was prepared, a series ofreactions, one cycle of 94° C. for one minute, 30 cycles of 94° C. for30 seconds-55° C. for 1 minute-72° C. for 3 minutes, and one cycle of72° C. for 10 minutes were carried out. An aliquot of this reactionmixture was subjected to agarose gel electrophoresis and an amplifiedabout 1.8 kb fragment was observed. This amplified fragment was digestedwith HindIII and SacI, and the about 1.5 kb DNA fragment produced wasrecovered from the gel after agarose gel electrophoresis. TheHindIII-SacI digested plasmid vector pUC119 was mixed with the aboveabout 1.5 kb DNA fragment to allow to ligate, followed by introductioninto Escherichia coli JM109. The plasmid harboured by the resultingtransformant was examined, the plasmid with only one molecule of the 1.5kb fragment inserted was selected and designated the plasmid pTC4.

FIG. 8 shows a restriction map of the plasmid pTC4.

(10) Determination of Nucleotide Sequence of DNA Fragment derived fromThermococcus celer Contained in Plasmid pTC4 and Protease TCES Gene

In order to determine the nucleotide sequence of a protease gene derivedfrom Thermococcus celer inserted into the plasmid pTC4, the deletionmutants wherein the DNA fragment portion inserted into the plasmid hadbeen deleted in various length were prepared using Kilo SequenceDeletion Kit. Among them, several mutants having suitable length ofdeletion were selected and the nucleotide sequence of each of theinserted DNA fragment parts was determined by the dideoxy method, andthese results were combined to determine the nucleotide sequence of theinserted DNA fragment contained in the plasmid pTCS4. SEQ ID No. 15 ofthe Sequence Listing shows the resulting nucleotide sequence.

By combining the sequence with the nucleotide sequence of the insertedDNA fragment contained in the plasmid pTCS6 obtained in Example 1-(8),the whole nucleotide sequence of the protease gene derived fromThermococcus celer was determined. SEQ ID No. 1 and 2 of the SequenceListing show the nucleotide sequence of open reading frame present inthe nucleotide sequence and the amino acid sequence deduced therefrom ofthe protease derived from Thermococcus celer, respectively. The proteasederived from Thermococcus celer encoded by the gene was designated theprotease TCES.

(11) Preparation of Plasmid pBTC6 Containing Protease TCES Gene

The plasmid pTCS6 was digested with HindIII and SspI (manufactured byTakara Shuzo Co., Ltd.), which was subjected to 1% agarose gelelectrophoresis to recover the separated about 1.8 kb DNA fragment.Then, the plasmid vector pBT322 (manufactured by Takara Shuzo Co., Ltd.)was digested with HindIII and EcoRV, which was mixed with the about 1.8kb DNA fragment to allow to ligate, followed by introduction intoEscherichia coli JM109. Plasmids were prepared from the resultingtransformant, the plasmid containing only one molecule of the 1.8 kbfragment was selected and designated the plasmid pBTC5.

Then, the plasmid pBTC5 was completely digested with HindIII and KpnI,which was blunt-ended and was subjected to intramolecular ligation,followed by

introduction into Escherichia coli JM109. Plasmids were prepared fromthe resulting transformant, and the plasmid from which the above tworestriction enzyme sites had been removed was selected and designatedthe plasmid pBTC5HK.

Further, the plasmid pBTC5HK was digested with BamHI, which wasblunt-ended, and was subjected to intramolecular ligation, followed byintroduction into Escherichia coli JM109. Plasmids were prepared fromthe resulting transformant, the plasmid from which the BamHI site hadbeen removed was selected and designated the plasmid pBTC5HKB.

The primer TCE12 which can introduce the EcoRI site and the BamHI sitein front of an initiation codon on the protease TCES gene, and theprimer TCE20R which has 16 bp-long nucleotide sequence complementary toa 3′ part of the SacI site of the plasmid pTCS6 and can introduce theClaI site and a termination codon were synthesized. SEQ ID Nos. 18 and19 of the Sequence Listing show the nucleotide sequences of the primerTCE12 and the primer TCE20R, respectively. A PCR reaction mixture wasprepared using these two primers and using a chromosomal DNA of

Thermococcus celer as a template. A reaction of 25 cycles, each cycleconsisting of 94° C. for 30 seconds-55° C. for 1 minute-72° C. for 1minute, was carried out to amplify an about 0.9 kb DNA fragment havingthese two oligonucleotides on both ends and containing a part of theprotease TCES gene.

The above about 0.9 kb DNA fragment was digested with EcoRI and ClaI(manufactured by Takara Shuzo Co., Ltd.), which was mixed with theEcoRI-ClaI digested plasmid pBTC5HKB to allow to ligate, followed byintroduction into Escherichia coli JM109. Plasmids were prepared fromthe resulting transformant, and the plasmid containing only one moleculeof the about 0.9 kb fragment was selected and designated the plasmidpBTC6.

(12) Preparation of Plasmid pTC12 Containing Protease TCES Gene

The plasmid pBTC6 was digested with BamHI and SphI, which was subjectedto 1% agarose gel electrophoresis to recover the separated about 3 kbDNA fragment. Then, the plasmid pUC-P43SD where the ribosome bindingsite sequence derived from Bacillus subtilis P43 promoter was introducedbetween the KpnI site and the BamHI site of the plasmid vector pUC18(manufactured by Takara Shuzo Co., Ltd.) (the nucleotide sequence of thesynthetic oligonucleotides BS1 and BS2 used for introduction of thesequence are shown in SEQ ID Nos. 20 and 21 of the Sequence Listing) wasdigested with BamHI and SphI, which was mixed with the previouslyrecovered about 3 kb DNA fragment to allow to ligate, followed byintroduction into Escherichia coli JM109. Plasmids were prepared fromthe resulting transformant, the plasmid containing only one molecule ofthe above about 3 kb DNA fragment was selected and designated theplasmid pTC12.

(13) Preparation of Plasmid pSTC3 Containing Protease TCES Gene forTransforming Bacillus subtilis

The above plasmid pTC12 was digested with KpnI and SphI, which wassubjected to 1% agarose electrophoresis to recover the separated about 3kb DNA fragment. Then, the plasmid vector pUB18-P43 was digested withSacI, which was bunt-ended and allowed to self-ligate to give theplasmid vector pUB18-P43S from which the SacI site had been removed.This was digested with KpnI and SphI, which was mixed with thepreviously recovered about 3 kb DNA fragment and allowed to ligate,followed by introduction into Bacillus subtilis DB104. Plasmids wereprepared from the resulting kanamycin-resistant transformant, and theplasmid containing only one molecule of the above about 3 kb DNAfragment was selected and designated the plasmid pSTC2.

Then, the plasmid pSTC2 was digested with SacI and was subjected tointramolecular ligation, followed by introduction into Bacillus subtilisDB104. Plasmids were prepared from the resulting kanamycin-resistanttransformant, the plasmid containing only one SacI site and designatedthe plasmid pSTC3.

Then, Bacillus subtilis DB104 harbouring the plasmid pSTC3 wasdesignated Bacillus subtilis DB104/pSTC3.

FIG. 10 shows a restriction map of the plasmid pSTC3.

Example 2

(1) Preparation of Chromosomal DNA of Pyrococcus furiosus

Pyrococcus furiosus DMS3638 was cultured as follows. A medium having thecomposition of 1% trypton, 0.5% yeast extract, 1% soluble starch, 3.5%Jamarin SÅSolid (manufactured by Jamarin Laboratory), 0.5% JamarinSÅLiquid (manufactured by Jamarin Laboratory), 0.003% MgSO₄, 0.001%NaCl, 0.0001% FeSO₄ Å7H₂O, 0.0001% CoSO₄, 0.0001% CaCl₂ Å7H₂O, 0.0001%ZnSO₄, 0.1 ppm CuSO₄ Å5H₂O, 0.1 ppm H₃BO₃, 0.1 ppm KAl (SO₄)₂, 0.1 ppmNa₂MoO₄ Å2H₂O, 0.25 ppm NiCl₂ ÅH₂O was placed in a 2 liter mediumbottle, and was sterilized at 120° C. for 20 minutes, nitrogen gas wasblown into the medium to purge out the dissolved oxygen, and the abovebacterial strain was inoculated into the medium, followed by subjectingto stationarily culture at 95° C. for 16 hours. After the completion ofcultivation, the cells were collected by centrifugation.

Then, the resulting cells were suspended in 4 ml of 50 mM Tris-HCl (pH8.0) containing 25% sucrose, to this suspension was added 2 ml of 0.2 MEDTA and 0.8 ml of lysozyme (5 mg/ml) and incubated at 20° C. for 1hour, 24 ml of a SET solution (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl,pH 8.0), 4 ml of 5% SDS and 400 μl of proteinase K (10 mg/ml) were addedthereto and incubated at 37° C. for another 1 hour. The reaction wasstopped by extraction with phenol-chloroform, followed by ethanolprecipitation to obtain about 3.2 mg of the chromosomal DNA.

(2) Genomic Southern Hybridization of Pyrococcus furiosus ChromosomalDNA

A chromosomal DNA of Pyrococcus furiosus was digested with SacI, NotI,XbaI, EcoRI and XhoI (all manufactured by Takara Shuzo Co., Ltd.),respectively. An aliquot of the reaction mixture was further digestedwith SacI and EcoRI, which was subjected to 1% agarose gelelectrophoresis, followed by southern hybridization according to theprocedures described in Example 1-(5). A ³²P-labeled DNA, which wasprepared using an about 0.3 kb DNA fragment obtained by digesting theabove plasmid p1F-2R(2) with EcoRI and PstI as a template and usingBcaBEST DNA Labeling kit (manufacture by Takara Shuzo Co., Ltd.) and[α-³²P]dCTP, was used as a probe. A membrane was washed in 2×SSCcontaining SDS to the final concentration of 0.5% at room temperature,rinsed with 2×SSC and the autoradiogram was obtained. As a result, asignal was observed in two DNA fragments of about 5.4 kb and about 3.0kb produced by digesting a Pyrococcus furiosus chromosomal DNA with SacIand it was indicated that a protease gene was present on respectivefragments. When the SacI-digested fragment was further digested withSpeI (manufactured by Takara Shuzo Co., Ltd.), the signal of the aboveabout 5.4 kb fragment did not show the change but the signal which hadbeen seen in the about 3.0 kb fragment was lost, and a signal was newlyobserved in the about 0.6 kb fragment. Since the SpeI site is notpresent in the protease PFUL gene represented by SEQ ID No. 7 of theSequence Listing, it was suggested that a signal on the about 0.6 kbfragment obtained by the digestion with SacI and SpeI was derived from anovel hyperthermostable protease (hereinafter referred to as “proteasePFUS”). In addition, regarding the products from digestion of Pyrococcusfuriosus chromosomal DNA with XbaI, a signal was observed on two DNAfragments of about 3.3 kb and about 9.0 kb. From a restriction map ofprotease PFUL gene shown in FIG. 1, it was presumed that the about 3.3kb fragment contained the protease PFUL gene and the about 9.0 kbfragment contained the protease PFUS gene. When the above chromosomalDNA was digested with XbaI and SacI, a signal was observed on the about2.0 kb fragment and the about 3.0 kb fragment. From the positions of theSacI and XbaI cleavage sites present on the protease PFUL gene shown inSEQ ID No. 7 of the Sequence Listing, it was presumed that the proteasePFUL gene is present on the about 2.0 kb SacI-XbaI fragment. On theother hand, it was presumed that the protease PFUS gene was present onthe about 3.0 kb fragment. Combining with the results on the digestionwith SacI, it was shown that no XbaI site is present on the about 3.0 kbDNA fragment obtained by the digestion with SacI alone.

(3) Cloning of 0.6 kb SpeI-SacI Fragment Containing Protease PFUS Gene

A chromosomal DNA of Pyrococcus furiosus was digested with SacI andSpeI, which was subjected to 1% agarose gel electrophoresis to recoverthe DNA fragment corresponding to about 0.6 kb from the gel. Then, theplasmid pBluescript SK(−) (manufactured by Stratagene) was digested withSacI and SpeI, which was mixed with the about 0.6 kb DNA fragment toallow to ligate, followed by introduction into Escherichia coli JM109 toobtain the plasmid library containing the chromosomal DNA fragments.Transformed Escherichia coli JM109 was seeded on a plate to form thecolonies, and the produced colonies were transferred to a Hybond-N+membrane, which was incubated at 37° C. for about 2 hours on a new LBplate. This membrane was treated with 0.5N NaOH containing 1.5M NaCl,then with 0.5M Tris-HCl (pH 7.5) containing 1.5 M NaCl, washed with2×SSC, air dried and the plasmid DNA was fixed to the membrane byirradiating with ultraviolet rays on a UV transilluminator. Thismembrane was treated at 50° C. for 2 hours in a hybridization buffer,and transferred to the same buffer containing a ³²P-labeled DNA probeused for southern hybridization described in Example 2-(2), to hybridizeat 50° C. for 18 hours. After the completion of hybridization, themembrane was washed in 2×SSC containing 0.5% SDS at room temperature,and washed at 37° C. Further, the membrane was rinsed with 2×SSC, airdried, exposed to a X-ray film at −80° C. for 12 hours to obtain anautoradiogram. About 500 clones were screened and, as a result, 3 clonescontaining a protease gene were obtained. From a signal on theautoradiogram, the positions of these clones were examined and thecorresponding colonies on the plate used for transfer to the membranewere isolated in LB medium.

(4) Detection of Protease PFUS Gene by PCR

Oligonucleotides which used for detection of a hyperthermostableprotease gene by PCR as a probe were designed based on the nucleotidesequences encoding two regions having the high homology with the aminoacid sequences of alkaline serine proteases derived from the knownmicroorganisms in the protease PFUL gene. Based on the amino acidsequence of the protease PFUL represented by FIGS. 2 and 3, the primers1FP1, 1FP2, 2RP1 and 2RP2 were synthesized. SEQ ID Nos. 22, 23, 24 and25 of the Sequence Listing show the nucleotide sequences of theoligonucleotides 1FP1, 1FP2, 2RP1 and 2RP2.

PCR reaction mixtures containing the plasmids prepared from the abovethree clones as well as the oligonucleotides 1FP1 and 2RP1, or 1FP1 and2RP2, or 1FP2 and 2RP1, or 1FP2 and 2RP2 were prepared, and a 30 cyclereaction was carried out, each cycle consisting of 94° C. for 30seconds-37° C. for 2 minutes-72° C. for 1 minute. It was shown that,when aliquots of these reaction mixtures were subjected to agarose gelelectrophoresis, respectively, the amplification of an about 150 bp DNAfragment was observed in all the three above plasmids when used theprimers 1FP2 and 2RP2, indicating that a protease gene was present onthese plasmids.

One of the above three clones was selected and designated the plasmidpSS3.

(5) Determination of Nucleotide Sequence of Protease PFUS Gene Containedin Plasmid pSS3

The nucleotide sequence of the inserted DNA fragment in the plasmid wasdetermined by the dideoxy method using the plasmid pSS3 as a templateand using the primer M4 and the primer RV (both manufactured by TakaraShuzo Co., Ltd.). SEQ ID No. 26 of the Sequence Listing shows theresultant nucleotide sequence and the amino acid sequence which wasdeduced to be encoded by the nucleotide sequence. By comparing the aminoacid sequence with that of the protease PFUL, the protease TCES andsubtilisin, it was presumed that the DNA fragment inserted in theplasmid pSS3 encoded the amino acid sequence having the homology withthese proteases.

(6) Cloning of N-Terminal Coding Region and C-Terminal Coding Region ofProtease PFUS by Inverse PCR Method

In order to obtain genes encoding N-terminal amino acid sequence andC-terminal one of the protease PFUS, the inverse PCR was carried out. Aprimer used for the inverse PCR was synthesized based on the nucleotidesequence of the inserted DNA fragment in the plasmid pSS3. SEQ ID Nos.27, 28 and 29 of the Sequence Listing show the nucleotide sequences ofthe primers NPF-1, NPF-2 and NPR-3.

A chromosomal DNA of Pyrococcus furiosus was digested with SacI and XbaIand was subjected to intramolecular ligation. PCR mixtures containing analiquot of the ligation reaction mixture and the primers NPF-1 andNPR-3, or NPF-2 and NPR-3 were prepared and a 30 cycle reaction wascarried out, each cycle consisting of 94° C. for 30 seconds-67° C. for10 minutes. When an aliquot of this reaction mixture was subjected toagarose gel electrophoresis, an about 3 kb amplified fragment wasobserved in a case of the use of the primers NPF-2 and NPR-3. Thisamplified fragment was recovered from the agarose gel, and mixed withthe plasmid vector pT7BlueT (manufactured by Novagen) to allow toligate, followed by introduction into Escherichia coli JM109. Plasmidswere prepared from the resultant transformant, the plasmid containing anabout 3 kb fragment was selected and designated the plasmid pS322.

On the other hand, an about 9 kb amplified fragment was observed in acase of the use of the primers NPF-1 and NPR-3. This amplified fragmentwas recovered from the agarose gel, the DNA ends were made blunt using aDNA blunting kit, followed by further digestion with XbaI. This wasmixed with the plasmid vector pBluescript SK(−) digested with XbaI andHincII to allow to ligate, followed by introduction into Escherichiacoli JM109. Plasmids were prepared from the resulting transformant, theplasmid containing an about 5 kb DNA fragment was selected anddesignated the plasmid pSKX5.

(7) Sequencing of Nucleotide Sequence of Protease PFUS Gene Contained inPlasmid pS322 and pSKX5

The nucleotide sequence of a gene encoding a N-terminal region of theprotease PFUS was determined by the dideoxy method using the plasmidpS322 as a template and using the primer NPR-3. SEQ ID No. 30 of theSequence Listing shows a part of the resulting nucleotide sequence andthe amino acid sequence deduced to be encoded by the nucleotidesequence.

Further, the nucleotide sequence of a region corresponding to a 3′ partof the protease PFUS gene was determined by the dideoxy method using theplasmid pSKX5 as a template and using the primer RV. SEQ ID No. 31 ofthe Sequence Listing shows a part of the resulting nucleotide sequence.

(8) Synthesis of Primer Used for Amplification of Full Length ProteasePFUS Gene

Based on the nucleotide sequence obtained in Example 2-(7), a primerused for amplification of the full length of the protease PFUS gene wasdesigned. Based on the nucleotide sequence encoding a N-terminal part ofthe protease PFUS shown in SEQ ID No. 30 of the Sequence Listing, theprimer NPF-4 which can introduce BamHI site in front of an initiationcodon of the protease PFUS gene. SEQ ID No. 32 of the Sequence Listingshows the nucleotide sequence of the primer NPF-4. In addition, based onthe nucleotide sequence in the vicinity of a 3′ region of the proteasePFUS shown in SEQ ID No. 31 of the Sequence Listing, the primer NPR-4having a sequence complementary to the nucleotide sequence and a SphIsite was synthesized. SEQ ID No. 33 of the Sequence Listing shows thenucleotide sequence of the primer NPR-4.

(9) Preparation of Plasmid pSPT1 Containing Hybrid Gene of ProteaseDerived from Pyrococcus furiosus and Protease TCES, for Transformationof Bacillus subtilis

By using a LA PCR kit (manufactured by Takara Shuzo Co., Ltd.), a PCRreaction mixture (hereinafter a PCR reaction mixture prepared by using aLA PCR kit is referred to as “LA-PCR reaction mixture”) containing theprimers NPF-4 and NPR-4 and a chromosomal DNA of Pyrococcus furiosus,and a reaction of 30 cycles, each cycle consisting of 94° C. for 20seconds-55° C. for 1 minute-68° C. for 7 minutes, was carried out toamplify an about 6 kb DNA fragment having these two primers on both endsand containing the coding region of the protease PFUS gene.

The about 6 kb DNA fragment was digested with BamHI and SacI, which wassubjected to 1% agarose gel electrophoresis to recover the separatedabout 0.8 kb DNA fragment. This fragment was mixed with the plasmidpSTC3 digested with BamHI and SacI to allow to ligate, followed byintroduction into Bacillus subtilis DB104. Plasmids were prepared fromthe resultant kanamycin-resistant transformant, and the plasmidcontaining only one molecule of the above 0.8 kb fragment was selectedand designated the plasmid pSPT1.

Bacillus subtilis DB104 harboring the plasmid pSPT1 was designatedBacillus subtilis DB104/pSTP1.

FIG. 14 shows a restriction map of the plasmid pSPT1.

(10) Preparation of Plasmid pSNP1 Containing Protease PFUS Gene forTransformation of Bacillus subtilis

The about 6 kb DNA fragment amplified in Example 2-(9) was digested withSpeI and SphI, which was subjected to 1% agarose gel electrophoresis torecover the separated about 5.7 kb DNA fragment. This was mixed with theplasmid digested with SpeI and SphI to allow to ligate, followed byintroduction into Bacillus subtilis DB104. Plasmids were prepared fromthe resulting kanamycin-resistant transformant, and the plasmidcontaining only one molecule of the 5.7 kb fragment was selected anddesignated the plasmid pSNP1. Bacillus subtilis transformed with theplasmid pSNP1 was designated as Bacillus subtilis DB104/pSNP1.

FIG. 15 shows a restriction map of the plasmid pSNP1.

(11) Determination of Nucleotide Sequence of Protease PFUS GeneContained in Plasmid pSNP1

An about 6 kb DNA fragment containing a protease gene inserted into theplasmid pSNP1 was fragmented into appropriate size with a variety ofrestriction enzymes, and the fragments were subcloned into the plasmidvector pUC119 or pBluescript SK(−). The nucleotide sequence wasdetermined by the dideoxy method using the resulting recombinant plasmidas a template and using a commercially available universal primer.Regarding a part from which the fragments having appropriate size couldnot be obtained, the primer walking method was used utilizing thesynthetic primers. The nucleotide sequence of an open reading framepresent in the nucleotide sequence of the DNA fragment inserted into theplasmid pSNP1 thus determined, and the amino acid sequence of a proteasederived from Pyrococcus furiosus deduced from the nucleotide sequenceare shown in SEQ ID Nos. 34 and 35, respectively.

(12) Synthesis of Primer for Amplification of Protease PFUS Gene

In order to design a primer, which is used for amplification of the fulllength protease PFUS gene and hybridizes to a 3′ part of the gene, thenucleotide sequence of the 3′ part of the gene was determined. First, anabout 0.6 kb DNA fragment containing the 3′ region of the protease PFUSgene, obtained by digestion of the plasmid pSNP1 with BamHI, was ligatedwith the plasmid vector pUC119 which had been digested with BamHI anddephosphorylated with alkaline phosphatase. The resulting recombinantplasmid was designated the plasmid pSNPD and the nucleotide sequence ofa region corresponding to the 3′ part of the protease PFUS gene wasdetermined by the dideoxy method using this as a template. SEQ ID No. 38of the Sequence Listing shows the nucleotide sequence, from the BamHIsite to 80 bp upstream nucleotide, present in the region (the sequenceof the complementary chain). Then, based on the sequence, the primerNPM-1 which hybridizes to a 3′ part of the protease PFUS gene andcontains a SphI site was synthesized. SEQ ID No. 39 of the SequenceListing shows the nucleotide sequence of the primer NPM-1.

In addition, the primers mutRR and mutFR for elimination the BamHI siteswhich are present about 1.7 kb downstream from an initiation codonwithin the protease PFUS gene were synthesized. SEQ ID Nos. 40 and 41 ofthe Sequence Listing show the nucleotide sequences of the primers mutRRand mutFR, respectively.

(13) Preparation of Plasmid pPS1 Containing Full Length Protease PUFSGene

Two sets of LA-PCR reaction mixtures containing Pyrococcus furiosuschromosomal DNA as a template and a combination of the primers NPF-4 andmutRR or a combination of the primers mutFR and NPM-1 were prepared, anda reaction of 30 cycles, each cycle consisting of 94° C. for 30seconds-55° C. for 1 minute-68° C. for 3 minutes, was carried out. Whenagarose gel electrophoresis was carried out using an aliquot of thisreaction mixture, an about 1.8 kb DNA fragment was amplified in a caseof the use of the primer NPF-4 and mutRR, and an about 0.6 kb DNAfragment in a case of the use of the primers mutFR and NMP-1.

Each amplified DNA fragment from which the primers had been removed byusing SUPREC-02 (manufactured by Takara Shuzo Co., Ltd.) was preparedfrom the two set of the PCR mixture. A LA-PCR reaction mixturecontaining both of these amplified DNA fragments and not containing theprimers and LA Taq was prepared, which was used to carry out heatdenaturation at 94° C. for 10 minutes, followed by cooling to 30° C.over 30 minutes and maintaining at 30° C. for 15 minutes to form ahetero duplex. Then, to this reaction mixture, LA Taq was added and wasincubated at 72° C. for 3 minutes, the primers NPF-4 and NPM-1 wereadded thereto and a reaction of 25 cycles, each cycle consisting of 94°C. for 30 seconds-55° C. for 1 minute-68° C. for 3 minutes, was carriedout. Amplification of an about 2.4 kb DNA fragment was observed in thisreaction mixture.

The about 2.4 kb DNA fragment was digested with BamHI and SphI, thefragments were mixed with the plasmid pSNP1, described in Example2-(11), from which the full length protease PFUS gene had been removedpreviously by digestion with BamHI and SphI, to allow to ligate,followed by introduction into Bacillus subtilis DB104. Plasmids wereprepared from the resulting kanamycin-resistant transformant, and theplasmid with only one molecule of the about 2.4 kb fragment inserted wasselected and designated the plasmid pPS1. Bacillus subtilis DB104transformed with the plasmid DB104 was designated Bacillus subtilisDB104/pPS1.

FIG. 16 shows a restriction map of the plasmid pPS1.

(14) Amplification of DNA Fragment of a Region from the Promoter to theSignal Sequence of Subtilisin Gene

A primer for obtaining a region from promoter to signal sequence ofsubtilisin gene was synthesized. First, with reference to the nucleotidesequence of a promoter region of subtilisin gene described in J.Bacteriol., volume 171, page 2657-2665 (1989), the primer SUB4 whichhybridizes to a part upstream of the region and contains the EcoRI sitewas synthesized (SEQ ID No. 36 of the Sequence Listing shows thenucleotide sequence of the primer SUB4). Then, with reference to thenucleotide sequence of a region encoding subtilis in described in J.Bacteriol., volume 158, page 411-418 (1984), the primer BmR1 which canbe introduce the BamHI site just behind the signal sequence wassynthesized (SEQ ID No. 37 of the Sequence Listing shows the nucleotidesequence of the primer BmR1).

The plasmid pKWZ containing subtilisin gene described in J. Bacteriol.,volume 17, page 2657-2665 (1989) was used as a template to prepare a PCRreaction mixture containing the primers SUB4 and BmR1, and a reaction of30 cycles, each cycle consisting of 94° C. for 30 seconds-55° C. for 1minute-68° C. for 2 minutes, was carried out. Agarose gelelectrophoresis of an aliquot of this reaction mixture confirmedamplification of an about 0.3 kb DNA fragment.

(15) Preparation of Plasmid pNAPS1 Containing Protease PFUS Gene forTransformation of Bacillus subtilis

The about 0.3 kb DNA fragment was digested with EcoRI and BamHI, whichwas mixed with the plasmid pPS1, described in Example 2-(13), whichpreviously had been digested with EcoRI and BamHI to allow to ligate,followed by introduction into Bacillus subtilis DB104. Plasmids wereprepared from the resulting kanamycin-resistant transformant and theplasmid containing only one molecule of the about 0.3 kb fragment wasselected and designated the plasmid pNAPS1. In addition, Bacillussubtilis DB104 transformed with the plasmid pNAPS1 was designatedBacillus subtilis DB104/pNAPS1.

FIG. 17 shows a restriction map of the plasmid pNAPS1.

Example 3

(1) Preparation of Probe for Detecting Hyperthermostable Protease Gene

The plasmid pTPR12 containing the protease PFUL gene was digested withBalI and HincII (both manufactured by Takara Shuzo Co., Ltd.), which wassubjected to 1% agarose gel electrophoresis to recover the separatedabout 1 kb DNA fragment. A ³²P-labeled DNA probe was prepared using theDNA fragment as a template and using BcaBEST DNA labeling kit and[α-³²P]dCTP.

(2) Detection of Hyperthermostable Protease Gene Present inHyperthermophile Staphylothermus marinus and Thermobacteroidesproteoliticus

Chromosomal DNAs were prepared from each 10 ml of cultures ofStaphylothermus marinus DSM3639 and Thermobacteroides proteoliticusDSM5265 obtained from Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH according to the procedures described in Example1-(3). Both chromosomal DNAs were digested with EcoRI, PstI, HindIII,XbaI and SacI, respectively, which were subjected to 1% agarose gelelectrophoresis, followed by southern hybridization according to theprocedures described in Example 1-(5). As a probe, ³²P-labeled DNA probeprepared in Example 3-(1) was used. A membrane was washed at 37° C. in2×SSC finally containing 0.5% SDS, rinsed with 2×SSC, and theautoradiogram was obtained. From this autoradiogram, a signal wasrecognized in an about 4.8 kb DNA fragment in a case of Staphylothermusmarinus chromosomal DNA digested with PstI, and in an about 3.5 kb DNAfragment in a case of Thermobacteroides proteoliticus chromosomal DNAdigested with XbaI, thus, indicating that a hyperthermostable proteasegene which hybridizes with the protease PFUL gene was present in theStaphylothermus marinus and Thermobacteroides proteoliticus chromosomalDNA.

Example 4

(1) Preparation of Crude Enzyme Preparation of Protease PFUS and TCES

Bacillus subtilis DB104 in which the plasmid pSTC3 containing thehyperthermostable protease gene of the present invention had beenintroduced (Bacillus subtilis DB104/pSTC3) was cultured in 5 ml of LBmedium (trypton 10 g/liter, yeast extract 5 g/liter, NaCl 5 g/liter, pH7.2) containing 10 μg/ml kanamycin at 37° C. for 8 hours. 250 ml of thesimilar medium was prepared in 1 liter Erlenmeyer flask, which wasinoculated with 5 ml of the above culture to culture at 37° C. for 16hours. Ammonium sulfate was added to a supernatant obtained bycentrifugation of the culture to 75% saturation, and the resultedprecipitates were recovered by centrifugation. The recoveredprecipitates were suspended in 4 ml of 20 mM Tris-HCl, pH 7.5, which wasdialyzed against the same buffer, and the resulting dialysate was usedas crude enzyme preparation (enzyme preparation TC-3).

Crude enzyme preparations were prepared from Bacillus subtilis DB104 inwhich the plasmid pSNP1 containing the hyperthermostable protease geneof the present invention was introduced (Bacillus subtilis DB104/pSNP1)or Bacillus subtilis DB104 in which the plasmid pSPT1 containing thehyperthermostable protease of the present invention, according to theprocedures described above, and the preparations were designated NP-1and PT-1, respectively.

These enzyme preparations were used to examine the protease activity bythe enzyme activity detecting method using the SDS-polyacrylamide gelcontaining gelatin or by the other activity detecting methods.

(2) Preparation of Purified Enzyme Preparation of Protease PFUS

Two tubes containing 5 ml of LB medium containing 10 μl/ml kanamycinwere inoculated with Bacillus subtilis DB104 in which the plasmid pNAPS1containing the hyperthermostable protease gene of the present inventionobtained in Example 2-(18) was introduced (Bacillus subtilisDB104/pNAPS1), followed by cultivation at 37° C. for 7 hours withshaking. Six Erlenmeyer flasks of 500 ml volume, each containing 120 mlof the similar medium, were prepared, and each flask was inoculated with1 ml of the above culture, followed by cultivation at 37° C. for 17hours with shaking. The culture was centrifuged to obtain the cells anda culture supernatant.

The cells were suspended in 15 ml of 50 mM Tris-HCl, pH 7.5, and 30 mgof lysozyme (manufactured by Sigma) was added thereto, followed bydigestion at 37° C. for 1.5 hours. The digestion solution washeat-treated at 95° C. for 15 minutes, followed by centrifugation tocollect a supernatant. To 12 ml of the resulting supernatant was added 4ml of an saturated ammonium sulfate solution, which was filtrated using0.45 μm filter unit (Sterivex HV, manufactured by Millipore), and thefiltrate was loaded onto the POROS PH column (4.6 mm×150 mm:manufactured by PerSeptive Biosystems) equilibrated with 25 mM Tris-HCl,pH 7.5 containing ammonium sulfate at 25% saturation. The column waswashed with the buffer used for equilibration, the gradient elution wasperformed by lowering the concentration of ammonium sulfate from 25%saturation to 0% saturation and at the same time increasing the

concentration of acetonitrile from 0% to 20% to elute the PFUS protease,to obtain the purified enzyme preparation NAPS-1.

750 ml of the culture supernatant was dialyzed against 25 mM Tris-HCl,pH 8.0 and adsorbed onto Econo-Pack Q cartridge (manufactured by BioRad)equilibrated with the same buffer. Then, the adsorbed enzyme was elutedwith a linear gradient of 0 to 1.5 M NaCl. The resulting active fractionwas heat-treated at 95° C. for 1 hour, and an ⅓ volume of a saturatedammonium sulfate solution was added thereto. After the filtration wascarried out using a 0.45 μm filter unit (Sterivex HV), the filtrate wasloaded onto the POROS PH column (4.6 mm×150 mm) equilibrated with 25 mMTris-HCl, pH 7.5 containing ammonium sulfate at 25%

saturation. The PFUS protease absorbed onto the column was elutedaccording to the procedures as in the enzyme preparation NAPS-1 toobtain the purified enzyme preparation NAPS-1.

To an appropriate amount of the purified enzyme preparation NAPS-1 orNAPS-1S was added trichloroacetic acid to the final concentration of8.3% to precipitate the proteins in the enzyme preparation, which wererecovered by centrifugation. The recovered precipitated protein weredissolved in a distilled water, an ¼ amount of a sample buffer (50 mMTris-HCl, pH 7.5, 5% SDS, 5% 2-mercaptoethanol, 0.005% Bromophenol Blue,50% glycerol) was added thereto, which was treated at 100° C. for 5minutes and subjected to electrophoresis using 0.1% SDS-10%polyacrylamide gel. After run, the gel was stained in 2.5% CoomassieBrilliant Blue R-250, 25% ethanol, and 10% acetic acid for 30 minutes,transferred in 25% methanol, and 7% acetic acid and the excess dye wasremoved over 3 to 15 hours. Both enzyme preparations NAPS-1 and NAPS-1Sshowed a single band, and a molecular weight deduced from migrateddistance was about 4.5 kDa.

(3) Sequencing of N-Terminal of Mature Protease PUFS

The purified enzyme preparation NAPS-1 prepared in Example 4-(2) wassubjected to electrophoresis using 0.1% SDS-10% polyacrylamide gel, andthe proteins on the gel was blotted onto a PVDF membrane (manufacturedby Millipore) using Semidry Blotter (manufactured by Nihon Eido).Blotting was carried out according to a method described inElectrophoresis, volume 11, page 573-580 (1990). After blotting, themembrane was stained with a solution of 1% Coomassie Brilliant BlueR-250, in 50% methanol, and destained with a 60% methanol solution. Apart of the membrane which had been stained was cut off, followed bysequencing of the N-terminal amino acid sequence by the automated Edmandegradation using G1000A protein sequencer (manufactured by HewlettePackard). SEQ ID No. 42 shows the resultant N-terminal amino acidsequence.

1. An isolated polynucleotide encoding a hyperthermostable protease,which polynucleotide hybridizes to the nucleotide sequence of SEQ IDNO:2, wherein hybridization is carried out by incubating in 6×SSC at 50°C. and washing with 2×SSC at 37° C.
 2. A method for preparing ahyperthermostable protease, comprising culturing a transformantcontaining the polynucleotide of claim 1, and harvesting ahyperthermostable protease from the culture.
 3. An isolatedpolynucleotide encoding a hyperthermostable protease, whichpolynucleotide hybridizes to the nucleotide sequence of SEQ ID NO:6,wherein hybridization is carried out by incubating in 6×SSC at 50° C.and washing with 2×SSC at 37° C.
 4. A method for preparing ahyperthermostable protease, comprising culturing a transformantcontaining the polynucleotide of claim 3, and harvesting ahyperthermostable protease from the culture.